Nucleotide Excision Repair Factor XPC Ameliorates Prognosis by Increasing the Susceptibility of Human Colorectal Cancer to Chemotherapy and Ionizing Radiation

Nucleotide excision repair (NER) is a DNA damage repair mechanism in mammals, but the relationship between NER and human colorectal cancer (HRC) progression has not been clarified yet. In this study, the expression of the NER genes XPA, XPC, XPF, XPG, ERCC1, and XPD was measured in normal and cancerous human colorectal tissue. Among them, only the XPC gene expression was significantly increased in colorectal cancer tissue. To establish the role of XPC in colorectal cancer, small interference RNA (siRNA) targeting XPC was used to knockdown the expression of XPC in HRC cell lines. In addition, an expression vector plasmid containing the XPC cDNA was constructed and stably transfected into HRC cell lines to overexpress the XPC gene. Interestingly, MTT and apoptosis assay demonstrated that XPC gene overexpression significantly increased the susceptibility of HRC cell lines to cisplatin and X-ray radiation. In order to study the relationship between XPC expression and the progression of HRC, XPC expression was measured in 167 patients with colorectal cancer. The results showed that patients with high XPC expression had longer survival time. Cox regression analysis showed that high XPC expression might be a potential predictive factor for colorectal cancer. In conclusion, XPC plays a key role in the susceptibility of colorectal cancer to chemotherapy and ionizing radiation and is associated with a good patients' prognosis.

INTRODUCTION
DNA repair plays an important role in maintaining genome integrity in both normal and tumor cells (1,2). Since many chemotherapeutic agents cause DNA damage, the abnormal expression of DNA damage repair genes is closely related to tumor prognosis (3,4). Among various DNA repair pathways, nucleotide excision repair (NER) is the main defensive barrier against DNA damage (5,6) and a major repair system for chemo radiotherapy-induced DNA damage (7,8).
NER works via two pathways: global genome repair (GGR) and transcription-coupled repair (TCR) (9,10). GGR is involved in injury repair for any genomic sequence, which is important to prevent carcinogenesis. The most important TCR function is to repair the DNA damage of actively transcriptional chains, which may be associated with tumor chemosensitivity. Monger proteins, xeroderma pigmentosum complementation group C (XPC) and its accessory subunits have been identified as protein complexes involved in recognizing DNA lesions and consequent recruit of other repair proteins (11). XPC defects are linked with many cancer types (12)(13)(14). Besides XPC, xeroderma pigmentosum gene group A (XPA) is another important NER gene, which works at the intersecting step of GGR and TCR pathways (15). Neither GGR nor TCR is initiated in the absence of XPA. Xeroderma pigmentosum group F (XPF) combines with ERCC1 to form a dimer that works as a 5 ′ DNA endonuclease, and XPD and XPG work as a DNA ligase and a 3 ′ DNA endonuclease (16), respectively. Although the NER mechanism involves approximately 30 proteins, these proteins are involved in the initiation stage of NER, and are its core proteins. However, the precise role of these NER proteins in colorectal cancer remains unclear.
Colorectal cancer is the second-most and third-most common cancer in women and men, respectively (17). Surgery and chemotherapy or radiotherapy are the main treatments for colorectal cancer. The 5 year overall survival rate of patients with colorectal cancer is approximately 50-75%, but the 3 year survival rate of patients with stage III colon cancer is only around 60% (18-20). Resistance to chemotherapy or radiotherapy reduces the survival time of patients with colorectal cancer (21,22). The mechanism involved in the chemo radiotherapy resistance is not yet clear. Previous studies reported that the polymorphism of NER genes is associated with the prognosis of colorectal cancer (23)(24)(25)(26), but the relationship between NER and colorectal cancer drug resistance is still poorly documented.
In the present study, the expression of NER genes (XPC, XPA, XPG, XPF, ERCC1, and XPD) was measured in human colorectal cancer and the corresponding normal tissues. The role of these genes in colorectal cancer susceptibility to chemotherapy and radiotherapy was also evaluated. In addition, the expression of these genesin colorectal cancer tissues from167 patients was evaluated by the Kaplan-Meier method and Cox proportional hazards regression. Based on these analyses, our aim was to clarify the relationship between NER genes and colorectal cancer prognosis, to provide some evidences on the role of targeting differential genes in colorectal cancer clinical treatment.

Clinical Data and Specimen Collection
All patients recruited in the present study read and signed the informed consent form, and the study was approved by the Ethics Committee of PLA324 Hospital and Bethune International Peace Hospital.
The clinical trial was divided into two separate sections. The first section included 36 fresh colorectal cancer specimens and 14 specimens of adjacent normal tissues that were collected from patients (25 males and 11 females, average age 52.5 ± 16.6 years) recruited between October 2009 and May 2010 at the Department of General Surgery PLA324 hospital (Chongqing, China).Samples were snap frozen and stored in liquid nitrogen within 20 min after collection. All frozen tissues were confirmed as normal or cancerous by pathological examination. Specimens included 28 adenocarcinomas, 5 mucinous adenocarcinomas, and 3 mucinous adenocarcinomas complicated with adenocarcinoma. The number of high, moderate, and poor differentiated cases was 5, 18, and 13, respectively. All patients were diagnosed with stage II-III colorectal cancer by pathological examination. To avoid stromal contamination, samples were collected from the tumor center (greater than 1.5 cm), and adjacent tissues located 5 cm away from the colorectal cancer tissue were removed and considered as a normal control. The tumor absence was also confirmed by pathological examination(within 20 min). In this section, all 36 patients had tumors more than 12cm above the anal margin and none of them had neoadjuvant chemotherapy before operation.
The second section included 167 patients who were not the same as the 36 patients above. According to TNM system based on clinical and pathologic stage, all 167 patients were TNM stage III colorectal cancer(21 rectal cancer patients and 146 colon cancer patients), who did not receive neoadjuvant chemotherapy before operation, but received chemotherapy with oxaliplatin and 5-FU. Colorectal carcinoma tissues were collected from patients who underwent radical resection of colorectal cancer at the Department of General Surgery PLA324 hospital and Bethune International Peace Hospital. The tissue was preserved in 4% formaldehyde and was embedded in paraffin, and included 127 adenocarcinomas, 26 mucinous adenocarcinomas, and 14 mucinous adenocarcinomas complicated with adenocarcinoma. The number of high, moderate, and poor differentiated cases was 55, 53, and 58, respectively. Patients were followed for 60 months using hospital records and by telephone interviews with patients or families. Only colorectal cancer related deaths were considered to perform survival analysis. We obtained a complete follow-up on 126 men and 41 women. At the end of the study, 91 patients were cancer-free, 35 had tumor recurrence and 76 died because of cancer metastasis.

Real-Time PCR
Relative gene expression was measured in triplicate in the patients of the first section using TaqMan gene expression assay(Applied Biosystems) and 7500 real-time PCR system for the following gene transcripts: XPA (Invitrogen, Hs00166045_m1), XPC (Invitrogen, Hs01104206_m1), XPD (Applied Biosystems, Hs00361161), XPF (Applied Biosystems, Hs00193342), XPG (Applied Biosystems, Hs00164482), and ERCC1 (Applied Biosystems, Hs01012161). Gene expression was normalized to β-actin. To ensure that β-actin itself did not change between different samples, the ratio between β-actin and GAPDH, a second reference gene, was calculated. Relative gene expression was estimated using the 2 − CT method as previously described (27).

Western Blot
Approximately 100 mg of colorectal tissue from the patients of the first section was homogenized in a glass homogenizer and lysed using 1 ml of precooled RIPA lysate for 15 s prior to incubation in an ice bath for 10 min. Cells were disrupted using ultrasounds (100 W for 5 s), centrifuged at 12,000 × g at 4 • C for 10 min, and the supernatants were harvested. Total protein concentration was quantified using the Bradford method.
Fifty micrograms of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel, and proteins were transferred onto a polyvinylidene fluoride (PVDF) transfer membrane. The membrane was semidried at 20 V for 15 min, blocked with 5% skim milk for 4 h, washed three times of 5 min each with tris-buffered saline (TBS). Subsequently, goat anti-human XPC polyclonal antibody (Santa Cruz Biotechnology) (1:200) was added, and the membrane was incubated at 4 • C overnight. After incubation with the primary antibody, a HRP-conjugated rabbit anti-goat IgG (1:3,000) (Zhongshan Golden Bridge Biotechnology Co., Beijing, China) was added, and the membrane was incubated at room temperature for 2 h. The membrane was stained using an enhanced chemiluminescence (ECL) reagent (Pierce, USA) and imaged onto an X-ray film (Fuji film, Japan) by autoradiography. Quantity One Imagine System and analysis software (Bio-Rad, USA) were used to quantitatively analyze the specific strips. βactin was selected as the internal control. The relative protein level was expressed as the ratio between XPC and β-actin densities.

Plasmid Construction for SiRNA Targeting XPC in Colorectal Cancer Cells
An effective targeted versican sequence (5 ′ -GGATGAAGCCCTCAGCGAT-3 ′ ) was screened from GenBank (No. giNM_004628, available at www.pubmed.com). The oligonucleotide chains were designed as a template based on the base pairing rule.
The pSilencer TM 5.1-H1 Retro Vector (Ambion, No. AM5784) was digested using the restriction enzymes Hind III and BamH I followed by ligation with T4 DNA ligase. Then, the recombinant DNA was transformed into fresh competent E. Coli DH5α cells. The recombinant clones were picked from a solid Luria-Bertani (LB) broth plate containing 100 µg/ml ampicillin. The positive clones were confirmed by PCR and sent to the Shanghai GeneChem Company for sequencing. The confirmed efficient vector was called pSilencer TM 5.1-XPC siRNA, and the corresponding control vector was called pSilencer TM 5.1-XPC control. Lipofectamine TM 2000 was used to transfect SW1463 cells with the pSilencer TM 5.1-XPC siRNA and pSilencer TM 5.1-XPC control. Additional puromycin (1 µg/ml) was added to select the positive clones.

Stable Transfection of Colorectal Cancer Cells With the pcDNA3-XPC Plasmid
The pXPC-3 plasmid, carrying the XPC gene cDNA, was kindly donated by Junlei Zhang (Microbiology and Immunology Department of the third military medical university). A 3.4-kb DNA fragment containing the XPC gene cDNA was removed from the pXPC-3 plasmid DNA by Sfi I digestion and inserted into the Sfi I site of the pcDNA3.1(+) (Invitrogen) to obtain the pcDNA3-XPC plasmid. SW1463 cells and HCT116 cells were seeded in 100-mm cell culture dishes with 5 ml DMEM and cultured until a confluence of 70-80% was reached. Cells were transfected with pcDNA3-XPC plasmid DNA using the cationic lipid Lipofectamine R 2000 transfection reagent (10 µg plasmid DNA/50 µl Lipofectamine R 2000/100-mm dish) and incubated for 6 h. Cells were also transfected with pcDNA3 as a negative control using the same protocol.

Immunohistochemistry
Immunohistochemistry was performed according to procedures previously described (28).Tissue sections from patients of the clinical trial second section, 5 µm thick, were deparaffinized, rehydrated in graded alcohols, and processed using the streptavidin immunoperoxidase method. In brief, sections were subjected to antigen retrieval by microwave oven treatment for 10 min in 0.01 mol/L citrate buffer (pH 6.0). Slides were subsequently incubated in 10% normal serum for 30 min, followed by an overnight incubation at 4 • C with the appropriately diluted primary antibody. Mice anti-human monoclonal antibody was used at a 1:100 dilution. Subsequently, samples were incubated with biotinylated anti-mice or antirabbit immunoglobulins for 15 min at 37 • C, followed by streptavidin peroxidase complexes for 15 min at 37 • C. 3.3 ′diaminobenzidine was used as the chromogen, and hematoxylin was used as a nuclear counterstain.
Immunohistochemical evaluation was conducted by at least two independent observers that scored the estimated percentage of tumor cells showing nuclear staining, independently of signal strength. An arbitrarily defined 15% cutoff was used to classify the colorectal carcinoma data into categorical groups (positive vs. negative).

Cell Susceptibility Assay
SW1463 cells and HCT116 cells (1 × 10 6 /ml) were seeded in a 96-well plate (100 µl/well), and each treatment was performed FIGURE 2 | XPC protein expression in SW1463 and HCT116 cells prior to and after transfection with XPC siRNA or pcDNA3-XPC. (A) XPC protein expression by western blot in SW1463 and HCT116 cells. The top image shows a blot that was probed with XPC antibody, while the bottom image shows a blot that was probed with β-actin antibody. (B) XPC relative expression in SW1463 and HCT116 cells. XPC expression in the XPC silenced cells was significantly lower than that in the normal SW1463 and HCT116 cells (P < 0.01), whereas XPC expression in the pcDNA3-XPC-transfected cells was significantly higher than that in the normal SW1463 and HCT116 cells (Define the column with a "+" as baseline, the control group transferred empty plasmid into the cells) (P < 0.01).

Cell Apoptosis Assay by FACS
Annexin-V-FITC apoptosis assay kit was purchased from Baosai Biological Technology Co., Ltd. (Beijing, China). SW1463 cells and HCT116 cells were treated with IRat different doses (0, 1, 2, 4 Gy) or cisplatin at different concentrations (0, 5, 20, 40 umol/L) for 4 h prior to digestion with 0.1% trypsin. The cell suspension was centrifuged at 1,000 rpm for 5 min, the supernatant was removed and the cell precipitate was washed twice with PBS.
Next, 100 µlAnnexin-V-FITC was added to the cell precipitate and cells were incubated for 10-15 min at room temperature without light. Cells were centrifuged at 1,000 rpm for 5 min and washed once with PBS. Cell apoptosis was detected using a FACScan Flow Cytometer (Becton Dickinson, USA), and data were analyzed using CellQuest 3.0 software (Becton Dickinson, USA).

Statistical Analysis
SPSS 18.0 statistics software (SPSS Science, Chicago, USA) was used to perform the statistical analysis. All data are expressed as mean ± standard deviation. Each experiment was repeated at least three times, and all data represent the mean of at least three parallel samples. Analysis of variance (ANOVA) and Student's t-test were used to compare the differences among groups and between two groups, respectively. A contingency table was generated with the chi-square or Fisher exact probability test for immunohistochemistry data. The overall survival probability was calculated by the Kaplan-Meier method with statistical differences evaluated by the log rank test. The relative death risk of colorectal cancer was estimated by a multivariate Cox proportional hazard model. A P < 0.05 was considered statistically significant.

Both XPC mRNA and Protein Expression Were Significantly Upregulated in Colorectal Cancer Tissues
NER genes, such as XPA, XPC, XPG, XPF, XPD, and ERCC1, were measured in normal and cancerous human colorectal tissues belonging to the first section of the clinical FIGURE 3 | XPCoverexpression suppressed cell growth in the presence ofIR or cisplatin. IR and cisplatin growth inhibition was evaluated in pcDNA3-XPC transfected cells and XPC silenced cells. pcDNA3-XPC transfected cell were more susceptible to IR radiation or cisplatin compared to control cells and siRNA cells. (A) Control SW1463 cells, pcDNA3-XPC transfected SW1463 cells and XPC silenced SW1463 cells were exposed to cisplatin at different concentrations, followed by MTT analysis after 4 h. (B) Control HCT116 cells, pcDNA3-XPC transfected HCT116 cells and XPC silenced HCT116 cells were exposed to cisplatin at different concentrations, followed by MTT analysis after 4 h. (C) Control SW1463 cells, pcDNA3-XPC transfected SW1463 cells and XPC silenced SW1463 cells were exposed to IR at different intensities, followed by MTT analysis. (D) Control HCT116 cells, pcDNA3-XPC transfected HCT116 cells and XPC silenced HCT116 cells were exposed to IR at different intensities, followed by MTT analysis. trial and the results showed that only XPC mRNA level in the colorectal cancer tissues was significantly increased compared with the corresponding normal tissue (P < 0.01). No significant differences in XPA, XPD, XPG, XPF, ERCC1 mRNAs was found between colorectal cancer and normal tissues (Figure 1). XPC protein expression was also measured in normal andcolorectal cancer tissues. Western blot results revealed that XPC protein appeared at the predicted site of 120 kDa and its expression in the colorectal cancer tissue was significantly higher than its expression in the normal tissue (P < 0.05) (Figure 1).

Different Susceptibility of Colorectal Carcinoma Cells to IR or Cisplatin After XPC Knockdown or Overexpression
XPC siRNA significantly downregulated XPC protein expression in SW1463 and HCT116 colorectal cancer cell line, whereas XPC protein expression was significantly upregulated in the cells transfected with pcDNA3-XPC (Figure 2). IR or cisplatin significantly suppressed the growth of normal SW1463 and HCT116 cells (P < 0.05). Furthermore, XPC siRNA significantly reduced cell sensitivity to IR or cisplatin (P < 0.05) (Figure 3). However, pcDNA3-XPC-transfected cells became sensitive to cisplatin compared with siRNA cells under the same cisplatin treatment (P < 0.05). These results revealed an important role of XPC in the radiotherapeutic and chemotherapeutic susceptibility of colorectal cancer cells.

Different Apoptotic Rate After IROR Cisplatin Treatment in XPC Overexpressing or Silenced SW1463 and HCT116 Cells
The treatment with IR or cisplatin significantly increased the apoptosis of SW1463 and HCT116 cells (P < 0.01 and P < 0.05, respectively). Interestingly, the transfection with pcDNA3-XPC increased the percentage of apoptosis induced by cisplatin treatment or IR (P < 0.05).When SW1463 and HCT116 cells were transfected with XPC siRNA, the percentage of cell apoptosis was significantly decreased after cisplatin treatment (Figure 4). FACS results revealed that XPC overexpression promoted SW1463 and HCT116 apoptosis after cisplatin treatment or IR, whereas  XPC silencing suppressed apoptosis, demonstrating a role of XPC in the radiotherapeutic and chemotherapeutic resistance of colorectal cancer.

Correlation Between XPC Expression and Colorectal Cancer Development
The relationship between XPC expression and the development of colorectal cancer was analyzed in the second section of the clinical trial. XPC (+) expression was 73.2% in well differentiated cancer tissue, 62.3 and 46.6% in moderately differentiated and poor differentiated cancer tissue, respectively ( Table 1 and Figure 5). Statistical analysis on immunohistochemistry data suggested that XPC (+) expression was significantly higher in well differentiated cancer tissue than poor differentiated cancer tissue (P < 0.05) ( Table 1).

Correlation Between XPC Expression and 5
Year Survival Rate Figure 6 shows the Kaplan-Meier curves of XPC with the adjusted P-values. The 5 year survival rate of patients with high XPC expression was significantly higher than that of patients with low XPC expression. However, the degree of tumor differentiation could significantly affect patient's survival time.
Indeed, patients with high XPC expression had a significantly longer 5 year survival time than patients with low XPC expression in both well differentiated and moderately differentiated tumors  (P < 0.01). However, this difference was not clear in the low differentiated group. Among the clinicopathological variables, tumor grade resulted as a significant prognostic factor after univariate analysis ( Table 2). The multivariate analysis by Cox was performed, including all the variables significantly associated with survival at the univariate analysis and indicated that XPC expression was an independent factor predicting patients' outcome ( Table 3).

DISCUSSION
Our study found that XPC expression in colorectal cancer tissue was significantly higher than that in the normal tissue and XPC overexpression increased the susceptibility of cells to cisplatin and IR in an in vitro experiment. Therefore, our study preliminarily proved that XPC might play an important predictiverole in the progression of colorectal cancer, although the mechanism is not yet clear. Tumoroccurrence, development and meta stasisis a multistep, multi-link, multi-faceted, multi-molecular process involving the activation of oncogenes and the inactivation of tumor suppressor genes (29), and the same molecule may play a different role at different stages of the tumorigenesis. Our study confirmed that the expression of XPC was closely related with the degree of differentiation of colorectal cancer. In addition, the expression of XPC in the highly differentiated colorectal cancer tissues was markedly higher than that in the   (30) suggested that low XPC expression facilitates the accumulation of oxidative damage, which in turn can cause tumorigenesis. All these evidences suggest that XPC, as a DNA damage recognizer, may play a protective role in the development of carcinogenesis. However, once the tumor occurs, the DNA repair mechanism may lead to cancer drug resistance and poor prognosis by repairing the DNA damage induced by chemotherapeutic drugs. Fautrel et al. (31) found that the expression of XPC in hepatic carcinoma tissue was significantly higher than that in normal hepatic tissue, and they speculate that the high XPC expression may lead to drug resistance of the liver tumor by enhancing DNA repair.The role of XPC in different tumors may be related to the mutation of the p53 gene. Yang et al. (27) further demonstrated that low XPC expression contributes to p53 mutation in bladder cancer tissues, both promoting tumorigenesis. Some studies suggest that XPC can facilitate apoptosis in the presence of normal p53 and such a role was not observed in mutated p53 cells (32). Moreover, other studies revealed that NOX-1 overexpression enhances XPC ability to recognize damage in the absence of p53 (30).Furthermore, high XPC expression promotes apoptosis by inhibiting the expression of the antiapoptotic protein Cas-2S,no matter whether p53 was mutated or not. Our study found that high XPC expression could improve the prognosis of patients with colorectal cancer by enhancing apoptosis induced by chemotherapeutic drugs. However, the study of Lin LC et al. (33) indicates that p53 mutation rate in colorectal cancer is ≥50% and p53 mutation results in poor prognosis of colorectal cancer patients. Thus, the link between XPC expression and p53 mutation needs further study.
Our study also found that high XPC expression enhanced cell sensitivity to IR. This effect might be due to the fact that XPC is not only a NER core protein but it is also involved in several other biological pathways, such as cell apoptosis, cell cycle regulation, oxidative damage recognition and base excision repair (30,34,35). The high XPC expression in colorectal cancer might be involved in these mechanisms and leading to a better prognosis. IR always induces apoptosis by damaging double strand DNA. Despras et al. (35) showed that low XPC expression weakened the double-stranded DNA injury repair capability of cells, while XPC overexpression enhanced such capability. Shell et al. (36) observed that high XPC expression occurs not only in DNA damages that can be repaired by NER but also in other types of DNA damages (e.g., DNA double-strand damage and oxidative damage).
Our results showed that even when transferring empty plasmid into the cells, the expression of XPC in SW1463 cells and HCT116 cells slightly increased. Our hypothesis is that this phenomenon might be related to the DNA recognition function of XPC, and unknown genes sequences of the plasmid transfected into the cell might induce the expression of XPC.
According to these studies, XPC participates in the recognition of DNA damages induced by chemotherapeutic drugs and initiates the subsequent apoptosis (severe DNA damages) or repair (mild DNA damages). Furthermore, the expression and function of XPC is regulated by many factors including p53 and NOX-1. Therefore, the pro-apoptotic role of XPC overexpression in colorectal cancer tissues might be affected by a variety of factors (e.g., the dose of chemotherapeutic drugs and p53 mutation) during chemotherapy or radiotherapy, although this aspect needs to be further explored. However, XPC is involved in several processes including cell cycle regulation, cell apoptosis and DNA damage repair, which undoubtedly provides new evidences in the occurrence, development and multi-drug resistance of colorectal cancer.

AUTHOR CONTRIBUTIONS
JL and PY conceived and designed the experiments; L-BH, YC, and XH performed the experiments; X-DM contributed reagents, materials, and analysis tools.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Overexpression of KLF5 inhibits puromycin-induced apoptosis of podocytes

Diabetic nephropathy (DN) is one of the most common microvascular complications associated with diabetes mellitus (DM); the incidence has been predicted to reach 7.7% by 2030 on a global scale. Krüppel-like factor 5 (KLF5) is involved in numerous important biological processes; however, the potential effects of KLF5 on podocytes in patients with diabetic nephrotic (DN) have not yet been investigated. In the present study, synaptopodin expression in podocytes was investigated using an immunofluorescence assay. Following this, the proliferation of podocytes was investigated using an MTT assay. In addition, KLF5 was overexpressed in podocytes, and cell cycle arrest and apoptosis was subsequently investigated using flow cytometry. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were performed to detect the expression levels of genes involved in the cell cycle and apoptosis, and the extracellular signal-regulated protein kinase (ERK)/p38 mitogen-activated protein (MAP) kinase pathway. The results demonstrated that treatment with puromycin aminonucleoside (PAN) suppressed the proliferation of podocytes in a dose- and time-dependent manner, and overexpression of KLF5 induced cell cycle arrest of podocytes regulated by PAN. Furthermore, overexpression of KLF5 was revealed to have inhibited PAN-induced apoptosis of podocytes, and that overexpression of KLF5 suppressed the ERK/p38 MAP kinase pathway in podocytes induced by PAN. Therefore, the results of the present study suggested that KLF5 may represent a potential therapeutic target for treatment of patients with DN.

Introduction
Diabetic mellitus (DM) is a progressive disease and is usually associated with numerous complications (1)(2)(3)(4). The incidence of DM is predicted to reach 7.7% by 2030 on a global scale (5,6). Diabetic nephropathy (DN) is one of the most common microvascular complications associated with DM and has become the second leading cause of end-stage renal disease in China (7). Numerous studies have demonstrated that well-managed blood glucose, blood pressure and blood lipid levels, and medicinal application of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, may postpone disease progression (8,9). The exact mechanisms underlying the pathogenesis of DN remain unclear. A previous study revealed that the onset of DN may be pertinent to oxidative stress in living cells (10). Podocytes are considered to be the most critical component of glomerular permselectivity (11). Changes in podocyte morphology and dysfunction are closely associated with renal diseases (12,13). The apoptosis of podocytes has been regarded as an important factor in the progression of DN (14). Therefore, investigating the molecular mechanism associated with the inhibition of podocyte injury is important for the development of novel therapeutic strategies for the treatment of patients with DN.
Krüppel-like factors (KLFs) are a type of transcriptional regulatory factor. KLFs are associated with cell proliferation, migration, apoptosis and tissue remodeling (15,16). Furthermore, KLFs are associated with the development of numerous diseases, including cardiovascular disease and cancer (17)(18)(19). KLF5, a member of the KLF family, can be regulated via phosphorylation, acetylation and ubiquitination following translation (20,21). KLF5 is associated with hypertensive nephropathy and diabetic retinopathy (22). However, the effects of KLF5 on DN are not fully understood.
Puromycin aminonucleoside (PAN) may disrupt the morphology of podocytes, trigger the overproduction of reactive oxygen species and induce nephrosis. Therefore, PAN is frequently used to establish a nephropathy model (23,24). The aim of the present study was to investigate the effect of KLF5 on PAN-induced injury of podocytes and to determine the underlying molecular mechanism. Therefore, the results of the present study may further the understanding of molecular mechanisms associated with DN and provide a potential therapeutic target for DN.

Materials and methods
Cell culture and cell treatment. Podocytes (MPC-5 cells) were acquired from the Bena Culture Collection (Beijing, China). Podocytes were cultured in Ham Nutrient Mix F12-Dulbecco modified Eagle medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 1% penicillin-streptomycin G (Biochrom, Ltd., Cambridge, UK) and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Podocytes were seeded in 6-well plates at a concentration of 3x10 5  Cell viability assay. Treated podocytes (2x10 3 cells/well) were seeded into a 96-well plate. The cells were treated with PAN at different concentrations (0, 5, 10, 20, 40, 60, 80 and 100 µg/ml) and maintained at 37˚C for 0, 6, 12, 24 and 48 h time intervals. Following this, 20 µl of MTT solution was added into each well and incubated for 30 min. A total of 150 µl dimethylsulfoxide was subsequently was added into each well. Finally, a microplate reader was used to determine the absorbance at 490 nm. Treatment with 60 µg/ml PAN for 6 h was selected for subsequent analysis as this resulted in decreased cell viability (described below).
Cell transfection. Podocytes were plated in 6-well plates at a density of 1x10 6 cells per well. Cells were treated with PBS or 60 µg/ml PAN for 6 h at 37˚C. KLF5 overexpression (1 ug) or negative control plasmids (pcDNA 3.1; 1 µg) were transfected into podocytes using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 12 h following transfection, the cells were used for subsequent experimentation.
Flow cytometry. Podocytes were treated as described above. Cells (2x10 5 cells/well) were seeded into 24-well plates and incubated overnight at 37˚C. To investigate the cell cycle, cells were centrifuged at 1,000 x g for 5 min at 4˚C and subsequently fixed using 70% (v/v) ethanol for 1 h at 4˚C. Following this, cells were washed with PBS and then incubated with 500 µl propidium iodide (PI)/RNase buffer (BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 30 min. Finally, a FACS-Calibur flow cytometer (BD Biosciences) was used to investigate cell cycle distribution, and ModFit LT 2.0 software (Verity Software House, Inc., Topsham, ME, USA) was used to analyze the results. To investigate apoptosis, cells were washed with PBS and then re-suspended with 0.5 ml binding buffer containing 5 µl Annexin V-fluorescein isothiocyanate and PI double stain (BD Biosciences) for 20 min at room temperature in the dark. Following this, the apoptotic rate was determined using a FACS-Calibur flow cytometer (BD Biosciences) and ModFit LT 2.0 software.
Statistical analysis. All data were analyzed by SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) using one-way analysis of variance with Tukey's test. Data are presented as the mean ± standard deviation. All experiments were performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

Results
PAN inhibits the proliferation of podocytes. Initially, synaptopodin expression in podocytes was investigated using immunofluorescence. The results revealed that the expression of synaptopodin was positive in podocytes (Fig. 1A). To further investigate the effect of PAN on the proliferation ability of podocytes, podocytes were treated with PBS (control) or PAN (5, 10, 20, 40, 60, 80, and 100 µg/ml) for 0, 6, 12, 24 and 48 h time intervals. The results revealed that the viability of podocytes were markedly inhibited following treatment with PAN in a dose-and time-dependent manner ( Fig. 1B;  P<0.05). It was observed that the cell viability significantly decreased when the cells were treated with 60 µg/ml PAN for 6 h. Therefore, the present study selected 60 µg/ml PAN to treat the cells for 6 h.
KLF5 is overexpressed in transfected podocytes. To further investigate the potential role of KLF5 in podocytes, KLF5 was overexpressed in podocytes via transfection with pCDNA3.1-KLF5 plasmids. RT-qPCR and western blot assays were performed to determine the expression levels of KLF5. The results demonstrated that KLF5 expression was significantly increased in podocytes transfected with pCDNA3.1-KLF5 compared with the NC group ( Fig. 2; P<0.001), which suggested that transfection to establish the podocyte injury model was successful.
Overexpression of KLF5 inhibits cell cycle arrest of PAN-treated podocytes. The results revealed that the proportion of cells in the G1 phase was increased in the PAN group compared with the control group (Fig. 3A). The number of cells in the G1 phase was decreased in the KLF5+PAN group compared with the NC+PAN group (Fig. 3A, P<0.05). In addition, the results demonstrated that treatment with PAN promoted cell cycle arrest in podocytes (P<0.05); however, this effect was markedly attenuated following overexpression of KLF5 (Fig. 3A, P<0.05). In addition, the expression levels of cyclin D1 and c-myc were investigated via RT-qPCR and western blot assays, and the results revealed that cells treated with PAN exhibited significantly decreased expression levels of cyclin D1 and c-myc compared with the NC group (P<0.01), whereas cells overexpressing KLF5 exhibited significantly increased expression levels of cyclin D1 and c-myc compared with the NC + PAN group (Fig. 3B-D, P<0.05).
Overexpression of KLF5 suppresses the apoptosis of PAN-treated podocytes. Flow cytometry was performed to determine whether KLF5 has an important role in the apoptosis of podocytes. The results demonstrated that the apoptosis of podocytes was significantly increased in the PAN group compared with the NC group (P<0.001; Fig. 4A); however, this effect was significantly attenuated following overexpression of KLF5 (P<0.05; Fig. 4A). Furthermore, the expression levels of genes associated with apoptosis (Bax, Bcl-2, caspase-3, caspase-8 and caspase-9) were investigated using RT-qPCR and western blot assays. The results revealed that treatment with PAN significantly enhanced the expression levels of Bax, caspase-3, caspase-8 and caspase-9 exhibited by podocytes (P<0.01 and P<0.001; Fig. 4B and C); however, overexpression of KLF5 significantly attenuated this effect (P<0.05 and P<0.01; Fig. 4B and C). In addition, podocytes treated with PAN exhibited significantly decreased Bcl-2 expression, and overexpression of KLF5 significantly attenuated this effect (P<0.05, P<0.01 and P<0.001; Fig. 4B and C).
Overexpression of KLF5 suppresses the activation of ERK/p38 mitogen-activated protein (MAP) kinase pathway in PAN-treated podocytes. MAP kinases have important roles in numerous cellular functions and are regulated via independent upstream activation cascades (26). In the present study, the effects of KLF5 and PAN on the ERK/p38 MAP kinase pathway in podocytes were investigated. The protein levels of p-ERK1/2,  ERK1/2, p-p38 and p38 in podocytes were investigated using a western blot assay. The results revealed that treatment with PAN significantly enhanced ERK1/2 and p38 phosphorylation compared with the control group; however, overexpression of KLF5 significantly attenuated these effects (P<0.001; Fig. 5).

Discussion
DN is one of the most common complications associated with diabetes and has become the most frequent causative factor resulting in the development of end-stage renal disease (27).  The pathogenesis of DN arises from glucose and lipid metabolic disorders, abnormal hemodynamics and oxidative stress (28,29). The results of the present study demonstrated that podocyte injury is an important factor resulting in the development of proteinuria and glomerular sclerosis, and has significant effects on the pathogenesis of DN (30). Considering that synaptopodin is a podocyte marker (31), the expression levels of synaptopodin in podocytes was investigated in the present study, and the results revealed that synaptopodin was highly expressed in podocytes.
Podocytes and the slit diaphragm within foot processes are critical components of the glomerular filtration barrier (32). Associated molecules, including nephrin, podocin, CD2 associated protein and α-actinin-4 have important roles in the development of proteinuria (33). A human podocyte cell line was used in the present study to investigate podocyte injury in vitro. Furthermore, the present study established a podocyte injury model in vitro via administration of PAN. This model provided the possibility to further investigate molecular mechanisms associated with DN (34)(35)(36). The results revealed that treatment with PAN markedly inhibited the proliferation of podocytes in a dose-and time-dependent manner.
KLF-5 is an important member of the KLF protein family, and is located at chromosome 13q21 and encodes a 55 kDa protein that contains 457 amino acids (37). In normal tissue, KLF5 extensively regulates numerous cellular processes, such as proliferation, differentiation, movement, inflammation and pluripotency (38,39). However, the role of KLF5 in DN remains unclear. In the present study, the results revealed that PAN induced cell cycle arrest in podocytes; however, overexpression of KLF5 significantly attenuated this effect via upregulation of cyclin D1 and c-myc expression levels. In addition, a previous study demonstrated that KLF5 inhibited the cell cycle progression of vascular smooth muscle cells via activation of cyclin D1 (40). Furthermore, overexpression of KLF5 was revealed to significantly suppress PAN-mediated apoptosis of podocytes. At a molecular level, the results of the present study revealed that PAN enhanced the expression levels of Bax, caspase-3, caspase-8 and caspase-9; however, treatment with PAN significantly reduced Bcl-2 expression. Furthermore, overexpression of KLF5 significantly decreased Bax, caspase-3, caspase-8 and caspase-9 expression levels, whereas overexpression of KLF5 significantly increased Bcl-2 expression in PAN-treated podocytes. A previous study revealed that treatment with PAN induces apoptosis of glomerular podocytes (41). A further study demonstrated that the downregulation of KLF5 is associated with G1 phase cell cycle arrest (42). The results of the present study were therefore consistent with previous studies. Therefore, it was concluded that overexpression of KLF5 alleviates PAN-mediated podocyte injury.
MAP kinases are serine/threonine-specific protein kinases (43)(44)(45). p38 MAP kinase and ERK are factors of the MAP kinase protein family (46,47). The results of the present study suggested that treatment with PAN induced the phosphorylation of ERK1/2 and p38. Overexpression of KLF5 suppressed the phosphorylation of ERK/p38 MAP kinase in PAN-treated podocytes. These results suggested that overexpression of KLF5 protected podocytes from injury via inhibited activation of the ERK1/2 and p38 pathways. However, a previous study demonstrated that KLF5 may promote the activation of ERK1/2 in breast cancer cells (48). This discrepancy may be due to the use of different cells.
Limitations of the present study included that all experiments were performed in vitro, and that the exact mechanism underlying the regulation of KLF5 remains unclear. Thus, future studies should perform further in vivo investigations to determine the role of KLF5 in DN. In conclusion, the results of the present study demonstrated that PAN inhibited the proliferation of podocytes, and that overexpression of KLF5 attenuated PAN-induced cell cycle arrest and apoptosis of podocytes. Furthermore, the results demonstrated that overexpression of KLF5 suppressed the ERK/p38 MAP kinase pathway in PAN-treated podocytes. The present study revealed that a potential therapeutic strategy for the treatment of DN may comprise the upregulation of KLF5 expression.


The genetic determinants of renal allograft rejection

We thank Massart et al. for their comments (1) on our recently published large-scale genome wide association study of renal transplant outcomes (2), and we welcome the opportunity to examine their signals in more detail. The two recipient genetic loci highlighted in their paper (3), rs10765602 (gene annotation CCDC67) and rs7976329 (gene annotation PTPRO), were well imputed in our study (INFO>0.95) and neither reached genome-wide significance in our reported analyses (2). This article is protected by copyright. All rights reserved.

The genetic determinants of renal allograft rejection
To the Editor: We thank Massart et al for their comments 1 on our recently published large-scale genome-wide association study of renal transplant outcomes, 2 and we welcome the opportunity to examine their findings in more detail.
The 2 recipient genetic loci highlighted in their paper, 3 rs10765602 (gene annotation CCDC67) and rs7976329 (gene annotation PTPRO), were well imputed in our study (INFO>0.95) and neither reached genome-wide significance in our reported analyses. 2 To provide additional confidence, we have reanalyzed our data following reimputation to the 1000 Genomes phase 3 reference panel via the Sanger Imputation Service (www. imputation.sanger.ac.uk) using Eagle and the Positional Burrows-Wheeler Transform package. 4 Table 1 indicates that neither single nucleotide polymorphism (SNP) reaches a nominal level of statistical significance in either donor or recipient genome for our broader definition of acute rejection (any acute rejection event recorded in the first 12 months after transplantation).
The lack of replication signal in our study, despite greater numbers of cases, may be due to a number of factors. We agree with Massart et al that one reason may be the differences in phenotype definition. Our study was primarily designed and powered to detect genetic variation in donor and recipient genomes associated with long-term graft survival, as this is the key unmet medical need in clinical renal transplantation outcomes, with currently no effective therapeutic options. Our acute rejection phenotype was established from reported national registry-based outcomes and was not specific to acute T cell-mediated rejection, and thus signal attenuation may be responsible for the difference. However, we note that in our study the recipient minor allele frequency differences between cases and controls are less than 1%, indicating almost complete attenuation.
Alternatively, it is possible that the signals found by Ghisdal et al 3 are false positives. Even genome-wide significant signals can be false positives, and as the authors used a pooled-DNA design, and employed a permutationbased joint test of association and linkage disequilibrium to determine the significance of hits in their discovery phase, it is difficult to determine the combined (discovery + replication) association p-values for their SNPs. We believe that further data are needed to resolve this issue.
We agree that genetic variation outside the HLA region is an important consideration in seeking to understand the pathogenesis of long-term graft survival and potentially identifying novel therapeutic targets to reduce cumulative allograft loss over time. We look forward to working with already established international collaborations 5 to identify these genetic determinants of long-term graft survival for the benefit of our patients.

D I SCLOS U R E
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.  Neither SNP was found to be significantly associated with the acute rejection phenotype in our analysis, in either recipients or donors. The SNPs were imputed to the 1000 Genomes Phase 3 reference panel and analyzed using SNPTEST (frequentist 1, method score). The first 5 principal components, the recruitment site, recipient age, donor age, recipient sex, donor sex, and the total number of HLA mismatches (at A, B, and DR) were included as covariates. Both SNPs, in both recipients and donors, were imputed to a high quality, with an info score and average maximum posterior call of greater than 0.95. Test = recipient/donor to indicate whether recipient or donor genotypes were tested (null as missing /control indicates whether blank records were treated as missing data, or as controls); rsID, SNP identifier; alleleA, noneffect allele (coded as 0); alleleB, effect allele (coded as 1); cases/controls AA, number of case/controls who were homozygous for allele A; cases/controls AB, number of case/controls who heterozygous; cases/controls BB, number of case/controls who were homozygous for allele B; MAF, minor allele frequency (frequency of least common allele in the given dataset); beta, beta coefficient relating to the coded allele (alleleB); SE, standard error of beta coefficient; P, P-value.


extfieldsofstudy: Biology Medicine

text:
Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Introduction
Chromatin conformation capture (3C) has emerged as an exceptionally useful method to study the topology of chromatin in the nucleus 1 . The 3C variant Hi-C allows measuring the contact frequencies of all chromatin interactions that occur in the nucleus in a single experiment 2 . Application of Hi-C has played an important role in the discovery and characterization of many fundamental principles of chromatin organization, such as TADs, compartments, and loops 3,4,5 .
Studies of chromatin architecture in the context of developmental transitions and cell differentiation are increasingly used to unravel the mechanisms of gene regulation during these processes 6,7,8,9 . One of the model organisms of great interest is Drosophila melanogaster, whose development and genome are well characterized. However, few studies that investigate chromatin architecture in Drosophila outside of in vitro tissue culture settings have been conducted 10,11 . In embryos 16-18 h post fertilization, TADs and compartments reminiscent of similar structures in mammals were identified 10 , which raises the question of which role they are playing in gene regulation during Drosophila embryo development. Especially in the early stages of development, prior to gastrulation, such studies are technically challenging. Before gastrulation, Drosophila embryos undergo 13 synchronous nuclear divisions that proceed at an extremely rapid pace of 8-60 min per cycle 12,13 . In addition to this, the lack of visual features to distinguish the different stages make it difficult to obtain tightly staged embryo material in sufficient quantities.
In order to develop a protocol that allows studying chromatin architecture in early Drosophila development at nuclear cycle resolution, we combined two existing techniques: in situ Hi-C, which allows the generation of high resolution whole genome contact maps 5 , and embryo staging using a transgenic Drosophila line expressing a eGFP-PCNA transgene 13,14 . This transgene localizes to the nucleus during interphase and disperses throughout the syncytial blastoderm during mitosis. Using this property, it is possible to easily distinguish different stages by their nuclear density and mitotic embryos by the dispersion of the GFP signal.
Together, these techniques enable studying the three-dimensional structure of chromatin in high resolution from as few as 20 Drosophila embryos. This protocol includes the instructions for harvesting and sorting Drosophila embryos to obtain populations of embryos from a single nuclear division cycle. It further describes how the obtained embryos are used to perform in situ Hi-C. The end result is a nucleotide library suitable for sequencing on next generation sequencing machines. The resulting sequencing reads can then be processed into detailed chromatin interaction maps covering the entire Drosophila genome.
Change collection plates twice a day. 3. Remove plates containing embryos from the collection chamber in 30-60 min intervals. Smaller intervals result in fewer embryos, but tighter distribution of developmental stages. Collect from multiple cages in parallel so that ideally >200 eggs are laid every 30-60 min. 4. Store the plates at 25 °C until the embryos reach the desired age. For blastoderm stage embryos (nuclear cycle 14), incubate for approximately 2 h. 5. After 2 h of incubation, add tap water from a squirt bottle to the collection plate so that the entire surface is covered with water. Suspend embryos and yeast using a soft brush. 6. Pour resuspended embryos from the collection plate into an embryo collection basket (commercial cell strainers with 100 µm pore size or homemade baskets 17 work well), adding additional tap water from a squirt bottle, if necessary. At this stage, combine embryos from all plates that were collected in parallel. The pooled sample represents a single batch. 7. Wash embryos well by rinsing the basket with tap water from a squirt bottle for 30 s until all yeast residue is washed away. 8. Dechorionate embryos by placing the collection basket into a 2.5% sodium hypochlorite solution in water. Light agitation by swirling facilitates removal of the chorion. Continue until embryos are sufficiently hydrophobic so that they float at the surface of the solution when the basket is lifted out and submerged again, which should take ~1.75-2 min. Caution: Sodium hypochlorite is corrosive. Wear appropriate personal protective equipment. Solutions containing <10% sodium hypochlorite can usually be disposed in the sink, make sure to check the regulations of the host institute. 9. Remove the basket from the solution and rinse thoroughly with tap water from a squirt bottle until the bleach smell is no longer noticeable.

Embryo Fixation
NOTE: Optimal fixation conditions, primarily the concentration of detergent, formaldehyde and the duration of fixation, need to be empirically determined to fit the stage of the embryos. For stages around the syncytial blastoderm, a final concentration of 0.5% Triton X-100 and 1.8% formaldehyde in the aqueous phase work well. For later stages beyond embryo stage 9, further optimization of these parameters may be necessary. All solutions used during fixation and sorting should contain protease inhibitors.
1. Invert collection basket and place it over a 15 mL conical centrifugation tube. Flush embryos from the basket into the tube using a Pasteur pipette dispensing PBS-T (PBS, 0.5% Triton X-100). 2. Let embryos settle at the bottom and adjust the total volume to 2 mL with PBS-T. 3. Add 6 mL of heptane and 100 µL of 37% formaldehyde in water.
Caution: Heptane and formaldehyde are toxic when inhaled or after skin contact. Wear appropriate personal protective equipment and work in a fume hood. Waste containing heptane or formaldehyde has to be disposed separately according to the host institute's regulations. 4. After the addition of the formaldehyde, start a 15 min timer and vigorously shake the tube up and down for 1 min by hand. The aqueous and organic phase will combine to form a shampoo-like consistency. 5. Agitate on a rotatory mixer until 10 min after the addition of formaldehyde. 6. Centrifuge at 500 x g for 1 min at room temperature to collect embryos at the bottom of the tube. 7. Aspirate the entire shampoo-like liquid and discard it, taking care not to aspirate any embryos. Small remaining quantities of shampoo-like supernatant do not cause problems. 8. 15 min after the addition of formaldehyde, resuspend the embryos in 5 mL of PBS-T with 125 mM glycine to quench the formaldehyde. Mix vigorously by shaking up and down for 1 min. 9. Centrifuge at 500 x g at room temperature for 1 min and aspirate supernatant. 10. Wash embryos by resuspending them in 5 mL of ice-cold PBS-T. Let embryos settle and aspirate all supernatant. 11. Repeat the wash in step 2.10 two more times. 12. Keep embryos on ice until sorting. Usually, it is a good idea to collect 3-4 batches of fly embryos before proceeding to sorting. However, embryos should be sorted the same day. Extended storage on ice or in the fridge leads to altered embryo morphology.

Embryo Sorting
NOTE: Sorting can be done on any fluorescent stereo microscope equipped with a GFP filter at 60-80X magnification.
1. Using a 1,000 µL pipette, transfer a batch of approximately 100 embryos to a small glass vessel suitable for sorting, preferably of a dark color, and place it on ice. 2. Sort embryos by nuclear density and cell cycle status (Figure 1) by pushing desirable embryos into a separate pile using a needle or syringe tip.
6. Incubate at -80 °C for 15 min. Centrifuge at 20,000 x g at 4 °C for at least 30 min. The DNA pellet is often very small, almost invisible, and can only be spotted due to the blue color of GlycoBlue. 7. Remove supernatant very carefully, moving the pipette tip into the tube along the opposite wall from where the DNA pellet is located.
Small remaining droplets are often easily removed during this step and the following washes by pushing them out of the tubes using a P10 tip rather than pipetting them out. 8. Wash pellet by adding 800 µL of 70% ethanol. Mix by inverting and centrifuge at 20,000 x g at room temperature for 5 min. Repeat this wash at least once. 9. Remove all traces of ethanol and leave the tube standing with the lid open for up to 5 min to air-dry. Once no liquid is remaining, add 50 µL of 10 mM Tris-Cl pH 8.0. Repeatedly pipette the solution over the area on the wall of the tube where the pellet was located to solubilize the DNA. 10. Add 1 µL of 20 mg/mL RNase A, mix by flicking the tube, and incubate at 37 °C for 15 min to digest RNA. The sample can now be stored in the fridge overnight or frozen at -20 °C indefinitely. 11. Check the concentration of DNA using a fluorescent dye based assay according to the manufacturer's instructions. The total amount of DNA in the sample should be at least 10 ng, otherwise too little material is available for amplification and library complexity will likely be low. When this happens, the amount of starting material was probably not sufficient, or material was lost along the way, perhaps during lysis and precipitation. Rotate beads with the DNA sample at 20 rpm for 15 min. 5. Separate beads on a magnetic stand and discard supernatant. 6. Resuspend beads in 600 µL of 1x B&W + 0.1% Triton X-100, and incubate at 55 °C for 2 min, shaking at 1,000 rpm. After separation, discard supernatant. Repeat this wash once. 7. Wash beads once with 600 µL of 10 mM Tris-Cl pH 8.0, and discard supernatant after separation. 8. Resuspend beads in 50 µL of 10 mM Tris-Cl pH 8.0.

Sequencing Library Preparation
NOTE: All library steps are done using components from a commercial DNA library preparation kit (see Table of Materials). However, alternative kits or other reagents may be substituted. Precipitation tends to form in the library preparation agents during freezer storage. It is therefore important to make sure that all precipitation is dissolved before using the reagents.

Representative Results
Sorted embryo populations at nuclear cycle 12, 13, and 14 (corresponding to 1:30, 1:45, and 2:10 hours post fertilization, respectively 12 ) and 3-4 h post fertilization (hpf) were obtained according to the procedures described in the protocol. By taking pictures of the eGFP-PCNA signal of each sorted embryo batch, it is possible to document the precise stage and cell cycle status of every single embryo that is used in downstream experiments. Example pictures of embryos from sorted populations are shown in Figure 1B-E. The output of the in situ Hi-C protocol is a nucleotide library ready to be sequenced on next generation sequencing machines. For this purpose, a final library concentration of at least 2-4 nM is usually required. Using the recommended amounts of input material, this concentration is reliably achieved ( Table 1).
The expected size distribution of DNA fragments after size selection is between 300-600 bp, with a maximum at around 500 bp (Figure 2A), depending on the exact shearing and size selection parameters. For sequencing, we recommend paired-end reads of at least 75 bp length to minimize the number of unmappable restriction fragments in the genome. High-resolution maps with 1-2 kb bin size can be obtained from 400 million reads. We recommend sequencing multiple biological replicates at a lower depth of ~150 million reads each, instead of sequencing a single replicate at very high depth. This allows assessment of the biological variation and leads to a lower number of discarded reads due to PCR duplication. For visual representation, the replicates can be combined. Before committing to sequencing a sample at high depth, we recommend running samples using shallow sequencing (a few million reads per sample) to determine basic library quality parameters as in Figure 2B. Analysis of Hi-C data requires significant computational resources and bioinformatics expertise. As a rough overview, the paired reads are mapped independently to the reference genome, the resulting alignments are filtered for quality and orientation, then a matrix of contacts at a given bin resolution or fragment level can be generated from the filtered alignments. The contact matrix is the basis for all further downstream analysis exploring TADs, loops, and compartments. For the initial analysis of the sequencing reads, several bioinformatics pipelines are available that enable processing of raw reads into contact matrices without much specialized bioinformatics knowledge 18,19,20,21,22,23 . How further analysis is carried out depends largely on the exact biological question under study and might require significant experience in programming and scripting in R or Python. However, several tools and algorithms to call TADs are available 5,24,25,26,27,28 , as well as software to analyze and explore Hi-C data in the web browser and as stand-alone desktop applications 29,30,31,32 .
Once processed, the quality of the library can be determined using different metrics ( Figure 2B). First, the rate of PCR duplicates, which is the number of sequenced read pairs arising from the same original molecule, should be as low as possible to limit the amount of wasted sequence reads. However, even libraries with >40% PCR duplication can be processed into high-quality contact maps if the duplicates are filtered. Second, the rate of filtered reads due to their orientation, as described in 4 , should consistently be lower than 10% of aligned read pairs.
During pre-gastrular development of Drosophila between nuclear cycle 12 and 14, the nuclear architecture is drastically remodeled 33 (Figure   3). At nuclear cycle 12, few TADs are detected, and the overall distribution of contacts is very smooth without many discernable features. This is dramatically changed at nuclear cycle 13 and 14, when TADs are increasingly prominent and unspecific long-range contacts are depleted.   Table 1: List of representative sequencing library statistics. For each library in the list, the number of embryos that were used for its generation, the amount of total DNA before biotin pulldown and shearing measured by Qubit, the number of PCR cycles used for amplification, and the final concentration of the sequencing library after purification and size selection are indicated.

Discussion
The protocol presented here is very effective at generating high-quality maps of the chromatin architecture in early Drosophila embryos.
Compared to an earlier protocol 34 , the approach described here uses an up-to-date in situ Hi-C procedure 5 , resulting in quicker processing, higher resolution, and less reagent usage. The overall procedure including the in situ Hi-C protocol is expected to work on a wide range of stages and experimental systems besides Drosophila. Since the protocol has a low input requirement, it could also be used on isolated cell populations. In Drosophila, when using the protocol for embryos outside the range described here, some parameters, in particular the fixation of the material, might need to be adjusted. Since older embryos develop a highly impermeable cuticle, raising the concentration of formaldehyde and prolonging fixation may be appropriate. For collection of embryos at stages other than nuclear cycle 14, the incubation times of embryos at 25 °C in step 1.4 need to be adjusted as follows: nuclear cycle 12, 70 min; nuclear cycle 13, 90 min; 3-4 hpf, 3:30 h.
During the 13 cleavage divisions (stage 1-4), the nuclei density roughly doubles with each division. The nuclei can easily be identified by their bright GFP fluorescence. During mitosis, eGFP-PCNA is not located in the nucleus, and its signal is dispersed throughout the embryo. This feature makes identifying embryos that are undergoing a synchronous cleavage division possible. For studying chromatin conformation, these mitotic embryos are usually not desirable, since the mitotic organization of chromatin is drastically different than the interphase organization 35 .
It is possible to adapt the protocol to specifically select embryos undergoing a synchronous mitotic division. In this case, only embryos with dispersed, non-nuclear distribution of eGFP-PCNA should be kept, and all other embryos should be discarded. Since the nuclear density cannot be determined, alternative methods to stage embryos by their morphology viewed in transmitted light microscopy must be employed. Presence of pole cells and nuclei at the embryo periphery indicate that the embryo has completed at least nuclear cycle 9, whereas visible cellularization at the periphery indicates nuclear cycle 14 12 .
Hi-C experiments can be successfully performed using a wide selection of restriction enzymes 5 . Current approaches typically use enzymes that recognize either a 4-base sequence, such as MboI, or a 6-base recognition site, such as HindIII. The advantage of 4-base cutters over 6-base cutters is that they offer higher potential resolution, given enough sequencing depth, and a more even coverage of restriction sites across the genome. There is no clear advantage in choosing one 4-base cutter over another 5,23,36,37 . The two most commonly used enzymes, MboI and DpnII, both recognize the same GATC recognition site. DpnII is less sensitive to CpG methylation, which is of no concern in Drosophila. The protocol presented here can also be successfully completed using DpnII as a restriction enzyme. In section 4.2. restriction enzyme and buffer have to be adjusted for DpnII compatibility, according to the manufacturer's recommendations.
If the fragment size of the sequencing library deviates significantly from the range shown in Figure 2A, cluster formation during sequencing may be less efficient or fail completely. In this case, the size distribution after shearing should be checked and shearing parameters adjusted accordingly. Peaks in the distribution of DNA fragments of very small (<100 bp) or very large (>1,000 bp) sizes indicates problems with size selection, such as carry over of beads or supernatant that are supposed to be discarded. Often these libraries with small peaks at these undesirable sizes, such as the one pictured, are still sequenced successfully with only a minor decrease in clustering efficiency.
High rates of PCR duplication should be avoided because this drastically reduces the number of usable sequence reads. The rate of PCR duplicates is directly related to the amount of input material. Using more input therefore usually alleviates problems with PCR duplication.