Paper Title (use style: paper title) Characterizing Intracellular Localization and Chromatin Remodeling Role of CHD6 in Human Fetal Development and in Glioblastoma Noah Basri and Nadejda Tsankova Icahn School of Medicine at Mount Sinai Departments of Pathology and Neuroscience Friedman Brain Institute Abstract— CHD6, a member of the CHD family of DNA-binding, chromatin-modifying proteins, has been shown to be upregulated in glioblastoma. Using immunofluorescence the intracellular region to which CHD6 localizes can be determined. Through the use of chromatin Immunoprecipitation (ChIP), candidate genomic loci enriched for CHD6 can be determined. Immunofluorescence and ChIP results for CHD6 using a current protocol are presented. Notably, immunofluorescence of fetal germinal matrix and adult subventricular zone showed principally cytoplasmic localization of CHD6, contrary to expectation. CHD6 ChIP showed partial enrichment in certain genes of possible significance to glioblastoma in fetal tissue relative to the positive control. A higher sample size is needed to test and evaluate the significance of these findings. The results presented here reveal new insights into the changes in the epigenetic landscape contributing to glioblastoma and the manipulation of CHD6 expression could be considered for treating specific incidents of glioblastoma. I. INTRODUCTION The set of proteins that constitute epigenetic regulators include so-called ‘writers,’ ‘editors’ and ‘readers’ of DNA methylation or histone post-translational modifications5. Whereas ‘writers’ are responsible for de novo modifications to DNA or DNA-bound histone proteins and ‘editors’ are responsible for chemically altering or reverting the modification placed by ‘writers’, ‘readers’ modulate and mediate interaction between proteins and protein complexes, including the transcriptional machinery5. Dysfunction or deregulation of epigenetic ‘writers,’ ‘editors’ or ‘readers,’ is widely implicated in stem cell activation and tumorigenesis5. Among the epigenetic ‘readers’ is a subset of chromodomain-containing proteins, the Chromodomain Helicase DNA-binding, or CHD, family of chromatin modifiers1,4,5,8,12. The CHD proteins are distinguished from other chromodomain-containing chromatin readers by their tandem chromodomains1,4,5,8, which have been shown to interact with methylated lysine residues correlated with either repressed transcription or active transcription (H3K27me3 and H3K4me3)1,4,11,12. Within the larger family of CHD proteins, are three subfamilies distinguished by their varying functional or conserved domains. Class I CHD proteins contain CHD1 and CHD2. The Class I CHD proteins are the best characterized in terms of function and structure,4,8,12. Chromatin Immunoprecipitation (ChIP) experiments, which utilize antibodies to selectively precipitate proteins bound to specific genomic regions, have been conducted to elucidate the DNA regions enriched for CHD112. Class II CHD proteins are distinguished by their dual PHD fingers, or plant homeodomains, and are composed of CHD3, CHD4 and CHD51,4,8,12. Class III CHD proteins are composed of CHD6, CHD7, CHD8 and CHD91,4,8,12. They are distinguished from the other two classes by their SANT (Switching-defective protein 3, Adaptor 2, Nuclear-receptor corepressor, Transcription Factor III B) and BRK (Brahma and Kismet) domains, which lie close to the proteins’ C-terminus1,4,12. The precise function of the BRK domain is unknown4; however, it has been shown to interact with the transcriptional repressor CTCF1,8, potentially coordinating chromatin loops7. Of considerable interest in the third CHD class is the poorly characterized nucleosome-remodeling protein CHD6. Though its precise mechanism of action has not been established8,12, CHD6 has been reported to be upregulated in glioblastoma3,10. As a result, insight into the intracellular localization of CHD6 as well as its enrichment in genetic loci of interest to glioblastoma, such as Olig22, EGFR (Epidermal Growth Factor Receptor)2 and GFAP (Glial Fibrillary Acidic Protein)2 using immunofluorescence and ChIP would provide a more holistic understanding of CHD6 expression and the epigenetic landscape of glioblastoma pathology. Immunofluorescence and ChIP experiments were performed to characterize the intracellular localization and chromatin-remodeling role of CHD6. Notably, immunofluorescence of fetal germinal matrix and adult subventricular zone showed principally cytoplasmic localization of CHD6, contrary to expectation. Through the use of ChIP, candidate genomic loci enriched for CHD6 were elucidated. CHD6 ChIP showed partial enrichment at the promoter of EGFR, one gene of possible significance to glioblastoma2, in fetal and glioblastoma tissues, relative to a presumed positive control locus where CHD6 has been previously demonstrate to bind8. However, higher sample size is needed to test and evaluate the significance of these findings. II. METHODS A. Immunofluorescence Paraffinized tissue sections (4 micrometers) previously fixed in 10% formalin were deparaffinized in xylene. Sections were rehydrated gradually through washes in graded alcohols: wash in 100% ethanol twice for 10 minutes each, then 95%, 75%, 50% ethanol for 5 minutes each then deionized water for 5 minutes. Slides were boiled in citrate-based buffer (pH=6) for 5 minutes then cooled on bench top for 30 minutes. Specimen was blocked in blocking buffer for 60 minutes using 0.25% TritonX and 1% normal donkey serum. Cells were incubated with anti-GFAP (rat) and CHD6 (rabbit) primary antibodies overnight at a concentration of 1:100. Sections were washed 3 times in PBS. Sections were then incubated for 1 hour with fluorochrome-conjugated secondary antibodies. Secondary antibodies for Rat (pink fluorescence) and rabbit (green fluorescence) were used. Following three more PBS washes, specimens were incubated with DAPI nuclear stain (blue fluorescence) for 5 minutes. Sections were examined using confocal microscopy. B. ChIP and qPCR Analysis DNA binding of CHD6 was assessed via chromatin immunoprecipitation (ChIP) assays. Frozen post-mortem brain tissue or neurosurgical de-identified specimens (adult cortex n=3, fetal germinal matrix n=3, glioblastoma n=3) were minced manually using razor blades in a Petri dish. Minced tissue was suspended in 1400uL 1% formaldehyde fixative and rotated for 10 minutes at room temperature. Formaldehyde fixation was quenched with 93.3uL 2M glycine (125mM final concentration) and rotated for 5 minutes at room temperature. Chromatin was sheared to 150-600bp9 and subjected to immunoprecipitation with antibodies against CHD6 or IgG (rabbit)2,9 as negative control (12-370, Millipore, 5 mg) after pre-incubation with magnetic Dynabeads from rabbits (11203D, Invitrogen). Ten percent of the lysate was saved as “input” for normalization analysis. The chromatin-associated DNA was purified using the QIAquick PCR Purification Kit (28104, Qiagen, Valencia, CA) and quantified by qPCR (Stratagene Mx3000P) using primers designed within the EGFR promoter (EGFRprom), ZIC1, a reputed gene enriched for CHD6, Olig2, and B2M, a housekeeping gene (Appendix 1). The data was analyzed as percent input recovery relative to ZIC1, the presumed positive control. Melting curves were analyzed to ensure specificity of the primers. The efficiency for all primers was calculated, deemed appropriate, and accounted for in the analysis2. III. RESULTS A. CHD6 Localizes to the Cytoplasm in Fetal Germinal Matrix and Adult Subventricular Zone Immunofluorescence staining of fetal germinal matrix showed principally strong cytoplasmic localization of CHD6 (Figure 2). There was high cell abundance in the fetal tissue compared to the adult tissue, consistent with the findings by Samuelsen et.al6. CHD6 staining appeared comparatively stronger in the fetal germinal matrix than in adult subventricular zone. GFAP, stained in pink, was clearly expressed in the radial glia projecting out of the germinal matrix. Notably, CHD6’s principally cytoplasmic localization seemingly conflicts with its reputed function as a DNA-binding protein and its hypothesized nuclear localization12. However, strong cytoplasmic staining could have obscured nuclear staining, and ChIP studies indicate the presence of locus- specific nuclear CHD6 presence. Further experimentation on CHD6 is required to determine whether it is natively found in the nucleus or if it translocates to DNA in response to some stimulus, as is seen in other transcription factors14. Intriguingly, there is remarkable CHD6 signal seen in the ependymal layer of the subventricular zone (Figure 1), which has not been previously demonstrated. Previous ChIP experiments have shown enrichment of CHD6 in CF-PAC, a pancreatic adenocarcinoma cell line, and Caco2, a colon cancer cell line8. Interestingly, pancreatic ductal cells (from which pancreatic adenocarcinoma arises), enterocytes, and ependymal cells have simple columnar structure17,18. Enterocytes and ependymal cells also have a similar brush border on their apical surface17. Whether these commonalities are related to CHD6 expression remains to be verified. B. CHD6 may be enriched at the EGFR Promoter in Fetal Tissue and Glioblastoma ChIP was performed on adult Cortex (n=3), fetal germinal matrix (n=3) and Glioblastoma (n=3) tissue types to assess for in vivo binding of CHD6 at genomic loci of potential interest in fetal development and gliomagenesis. The enrichment of CHD6 in each of the genomic loci of interest was calculated as percent of input recovery, and it was normalized to percent input enrichment of CHD6 at ZIC1, the positive control locus (Figure 4). The expectation was to see enrichment of CHD6 at gene loci, which are dysfunctionally upregulated in Figure 2 Note the principally cytoplasmic localization of CHD6 and strong CHD6 signal. Fetal germinal matrix under the confocal microscope. GFAP fluoresces pink, CHD6 is green and DAPI is blue. Figure 1 Adult subventricular zone under the confocal microscope. Note cytoplasmic localization of CHD6 and robust CHD6 signal in the ependymal layer, as indicated by the white arrow. GFAP fluoresces pink, CHD6 is green and DAPI is blue. glioblastoma, such as at Olig2 and EGFR, since the chromodomains of CHD6 bind methylated lysine residues associated with active transcription11. This preliminary data reveal greatest CHD6 enrichment at the EGFR promoter in fetal and glioblastoma samples, and at the Olig2 promoter in fetal samples, relative to ZIC1. Unfortunately, significance cannot be established in this data since the qPCR amplification of IgG samples yielded more than one amplicon product. IV. DISCUSSION The immunofluorescence staining of both fetal germinal matrix and adult subventricular zone showed principally cytoplasmic localization of CHD6. This contradicts the hypothesis that CHD6 is strictly relegated to DNA-binding in the nucleus. One possible explanation for these results is that most CHD6 could natively reside in the cytoplasm but translocate to the nucleus in response to binding of a cofactor. Lathrop et. al. report that CHD6 precipitates in complex with the transcription factor Nrf2, which is trafficked to the nucleus in response to cellular oxidative stress13,14. Analysis of the primary amino acid sequence of CHD6 reveals several potential nuclear localization signals (NLS)19. One possible mechanism for CHD6’s counterintuitive intracellular localization could be that CHD6’s NLS sequences are obscured by protein folding but are subsequently revealed following binding of a cofactor. Upon the emergence of the NLS sequences, importin, the protein responsible for protein import into the nucleus, could shuttle CHD6 to the genetic loci where CHD6 exerts its action as a chromatin remodeler. Both the immunofluorescence and ChIP experiments relied on primary antibodies effective at recognizing CHD6. Prior to the immunofluorescence and ChIP, no experiments validating the antibody and proving its efficacy in precipitating CHD6 were performed. Furthermore, although no published literature has elaborated on the finding that CHD6 is enriched at ZIC1, ZIC1 was used as the positive control locus in the ChIP experiment. Sancho et. al., independently reported that ZIC1 would act as an effective positive control. In designing primers for ZIC1, the UCSC Genome Browser was consulted for candidate loci that could be enriched for CHD6. Candidate loci were determined using enrichment of transcription factor CTCF as proxy for CHD6 since Sancho et. al. reported CTCF acting in complex with CHD68. To date, there has been no brain tissue ChIP-seq data for CHD6 upon which to base ZIC1 as a positive control more accurately. Operating under the conjecture that CHD6 is enriched at ZIC1, based on the assertion by Sancho et. al. is an assumption in this experiment. Another limitation of the data is the lack of a negative control. Since CHD6 is a chromatin remodeler that colocalizes with RNA Polymerase II8, an effective negative control would be loci of constitutively heterochromatic regions of DNA, such as telomeres or centromeres. Since these loci are constitutively heterochromatic, they are not transcribed and therefore enrichment of RNA Polymerase II and CHD6 would be unlikely in those regions. The qPCR amplification of the IgG samples yielding more than one amplicon could suggest the presence of primer dimers on the melting curve due to very little DNA pulled down in the immunoprecipitation. As a result, the experiment must be repeated with higher sample size in order to establish significance and make a strong assertion based on the ChIP data. Future experiments will seek to titrate parameters for and effectuate a ChIP protocol using MNase, as opposed to sonication, for chromatin fragmentation. More ChIP experiments on adult cortical, fetal and glioblastoma tissue isolated from postmortem brain are needed to confirm or deny the results presented here. Also, immunofluorescence on epileptic brain specimens might shed further light on the question of CHD6’s translocation to the nucleus. Epileptic seizures are characterized by failure to reuptake glutamate released into the synapse, resulting in prolonged and uncontrolled neuronal depolarization15. This behavior is highly taxing on neurons and often results in a state of cellular Figure 3 Enrichment of CHD6 binding is represented as percent input, relative to ZIC1, the positive control locus. Loci assessed include, EGFR promoter (green), B2M (purple), and Olig2 (blue). E n ri ch m en t o f im m u n o p re ci p it at ed D N A o v er 2 0 % i n p u t oxidative stress called excitotoxicity16. Examining such oxidatively stressed tissue through immunofluorescence could enhance the understanding of CHD6’s intra cellular localization. Going forward, experiments should seek to elucidate the coordination between CHD6 and CTCF8 in the formation of loops within chromatin7. These loops signify the topological landscape of chromatin and are as impactful to transcription as the molecular epigenetic landscape of histones and DNA7. To further the body of knowledge around chromatin loops and a potential role for CHD6 in catalyzing their formation, co- immunoprecipitation (co-IP) of CHD6 should be performed. If CHD6 precipitates in complex with CTCF and cohesin, it can be determined whether CHD6 has a correlative role in coordinating chromatin loops. The above data provide evidence that suggests a role for CHD6 in binding chromatin at actively transcribed loci, such as the EGFR promoter or Olig2 in fetal cortical and glioblastoma tissue. The results presented here reveal new insights into the changes in the epigenetic landscape contributing to glioblastoma. Manipulation of CHD6 expression could be considered for treating specific indications of glioblastoma by decreasing transcription of inappropriately expressed genes such as Olig2 and EGFR; however, further research is needed to fully understand CHD6’s role in gliomagenesis. ACKNOWLEDGMENT I would like to thank Dr. Tsankova and Dr. Tome-Garcia whose reagents, support and assistance were critical to performing the immunofluorescence and ChIP. I would also like to thank Parsa Erfani for his expertise and guidance throughout the lysate preparation and statistical analysis. Thank you to Dr. Walsh for collaborating on the experimental design, providing the CHD6 antibody and reporting ZIC1 as a positive control for CHD6 ChIP. REFERENCES [1] Allen et. al. (2007) Solution structure of the BRK domains from CHD7. J. Mol. Biol. 371, 1135–1140. [2] Erfani, P., Tome-Garcia, J., Canoll, P., Doetsch, F., & Tsankova, N. (2015). 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Enterocyte Cytoskeleton: Its Structure and Function. Comprehensive Physiology. 209–221. [18] Bottin-Grapin, Anne. 2004. Ductal Cells of the Pancreas. International Journal of Biochemistry and Cell Biology. 504-510. [19] Kosugi S., Hasebe M., Tomita M., and Yanagawa H. (2009) Systematic identification of yeast cell cycle-dependent nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Natl. Acad. Sci. USA 106, 10171-10176. I. Introduction II. Methods A. Immunofluorescence B. ChIP and qPCR Analysis III. Results A. CHD6 Localizes to the Cytoplasm in Fetal Germinal Matrix and Adult Subventricular Zone B. CHD6 may be enriched at the EGFR Promoter in Fetal Tissue and Glioblastoma IV. Discussion Acknowledgment References