Characterization of the Contradictory Chromatin Signatures at the 3′ Exons of Zinc Finger Genes

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3′ exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3′ exons of ZNFs are promoters. We further characterized the histone modifications at the 3′ ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3′ exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3′ ZNF exon was removed from its natural genomic location, the isolated ZNF 3′ exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3′ exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3′ exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3′ exons may function to protect the genome from inappropriate recombination rather than to regulate transcription.


An#body valida#on for Blahnik et al.
The an#bodies that we used for each experiment are listed in Table S1. Specifically, we used the H3K9me3 an#body from Cell Signalling (catalog number 9754), the H3K9me3 an#body from Abcam (catalog number 8898), the H3K36me3 an#body from Abcam (catalog number 9050), and the H3K4me3 an#body from Cell Signaling Technology (catalog number 9751). All of these an#bodies have been used extensively by the ENCODE Consor#um and by the Roadmap Epigenome Mapping Centers. They are all very specific and do not crossreact with other modified histones. The specificity of each an#body is documented in the following pages. This an#body was also validated for the Farnham lab by dot blot by Bing Ren's lab as part of the REMC Consor#um.
Tri--Methyl--Histone H3 (Lys9) An#body specificity was determined by pep#de ELISA. The graph depicts the binding of the an#body to pre--coated tri--methyl histone H3 (Lys9) pep#de in the presence of increasing concentra#ons of various compe#tor pep#des. As shown, only the tri--methyl histone H3 (Lys9) pep#de competed away binding of the an#body. For more details, see h[p:// www.cellsignal.com/products/9754.html H3K9me3 Cell Signaling Technology 9754 Also, this an#body was validated by the Brad Bernstein group as part of the ENCODE Consor#um. See below for valida#on performed at the Broad Ins#tute Abcam Cat#: ab9050 Lot#395432 This an#body was validated by dot blot by Bing Ren's lab as part of the REMC Consor#um.

Farnham Lab REMC ChIP and Library Construction Protocol Preparation of cross-linked cells (Record cell/patient information, date of culture/collection, etc. for future reference)
1. The amount of cells needed for ChIP-seq will vary depending on the histone mark to be analyzed. In general between 1 and 5 ug are used per histone antibody. A minimum of 20 ug is required to analyze all 6 histone marks; this corresponds to 0.5 to 2 x 10^7 cells, depending on the cell type. For primary cells or tissues, the samples can be used immediately or snap frozen in liquid nitrogen and crosslinked at a later date. If using immediately (or after culturing for a few passages), in a chemical hood add formaldehyde (from the 37% stock bottle) directly to the collection media or culture media to a final concentration of 1%. If using frozen cells, in a chemical hood prepare a 1% formaldehyde solution in PBS and add directly to frozen cell pellet; resuspend the cell pellet by pipetting up and down.
2. Rotate the cells in a tightly closed tube (for adherent cells use a shaking platform) for 10 minutes at room temperature. Do not crosslink for longer periods since this may cause cells to form aggregates that do not sonicate efficiently.
3. Stop cross-linking reaction by adding glycine to a final concentration of 0.125 M. We use a 10X (1.25 M) stock solution. Continue to rock or stir at room temp for 5 minutes.
4. For cells crosslinked in suspension, centrifuge cells at 430 rcf for 5 min at 4ºC, discard solution, wash pellet twice with ice-cold 1xPBS (mix by pipetting, pellet cells by centrifugation at 430 rcf for 5 min at 4ºC and discard wash solution). For adherent cells, pour off media and rinse plates twice with ice-cold 1x PBS and pour off wash solution. Using a silicon stopper cut in half, scrape cells in residual PBS and transfer cells from the culture dish to a 15 ml conical tube on ice.
Centrifuge the crosslinked cells at 430 rcf for 5 min at 4ºC. It is important to carefully aspirate supernatants so as to not lose cells. Note: media containing formaldehyde should be treated as hazardous waste.
5. Cells may now be used immediately for a chromatin preparation or snap frozen in liquid nitrogen and stored at -80°C, then shipped on dry ice to the Farnham lab.

Determining chromatin size and chromatin quantification
1. Take an aliquot of your chromatin sample from step 6 above (typically we use chromatin from 1-2 x 10^5 cells).
2. Add ChIP elution buffer to a total volume of 100 µl, add 12 µl 5M NaCl, boil samples in water bath for 20 minutes to reverse cross-links.
3. Allow sample to cool down, add 1 µl DNase-free RNase, incubate 20 minutes at 37ºC. This step is important because the presence of RNA results in false estimation of chromatin size. 4. Purify DNA using a PCR purification kit, elute in 30 µl water. Measure chromatin concentration by NanoDrop and calculate the chromatin yield. 5. Run 1.5-2 µg of chromatin on a 1.2% agarose gel to visualize average size of chromatin.
If the chromatin is larger than ~600 bp, repeat steps 1-5 and adjust the sonication conditions (e.g add more pulses).

Chromatin Immunoprecipitation
[For best results, proceed to ChIP assays on the same day as chromatin is prepared]  3. Assess ChIP enrichments by quantitative PCR (qPCR) before proceeding to preparation of Solexa libraries.

ChIP confirmation
Enrichment of histone marks in the ChIP samples are determined by quantitative real-time PCR (qPCR). The input sample is diluted with EB to give a final concentration of 2 ng/µl and serves as a reference. Prepare a master reaction mix for each library with triplicate reactions per primer set.
Add extra reagents for 10% of the total number of reagents to account for loss of volume. Add 14 µl of reaction mix to each PCR reaction well. Add 2 µl primer mix to each well.
Recipe for one reaction: The enrichment is then calculated by comparing relative enrichment for the target and a negative control. This is accomplished by dividing the relative DNA amount of each sample for a target primer set by the corresponding value for a negative control primer set. The resulting quotient represents the fold enrichment.

REMC Library Construction Protocol
The library protocol is based on the Illumina Sample Preparation Kit for Genomic DNA with some modifications. This protocol describes the preparation of libraries of ChIP DNA compatible with the Illumina sequencing platforms. Libraries are prepared from the ChIP sample as well as matching input DNA from the same cell type Step 1: End-Repaire using "End-It DNA Repair Kit" from Epicentre (Cat# ER0720). This step ensures that all DNA fragments are converted to 5'-phosphorylated blunt-ended DNA.
A) The entire ChIP DNA volume is used. Combine and mix the following components in a siliconized eppendorf tube: Step2: Addition of 'A' base to 3' Ends *Before starting, make up stocks of 1mM dATP using NEB 100mM dATP. (e.g. add 5ul of 100mM dATP to 495ul sterile RNase DNase free Gibco water; then make 25uL aliquots and freeze at -20C. You will want to freeze aliquots and defrost them only once).
A) Combine and mix the following components in PCR tubes: 34ul DNA from Step1 5ul Klenow buffer = NEB Buffer 2 10ul 1 mM dATP (you will have to make this up) 1ul Klenow fragment (3'→5' exo from NEB Cat# M0212s; 5,000 U/ml) x ul sterile water to bring total reaction volume to 50 ul B) Incubate for 30 minutes at 37°C (use PCR machine for incubation period).
C) Purify DNA using the MinElute PCR Purification Kit and protocol, and eluting in 11ul of EB.
Step 3: Adapter Ligation *Dilute Illumina adapters 1:10 with water to adjust for the smaller quantity of DNA. Excess adapters can interfere with sequencing. Use diluted primers for one week and then discard.

5' P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
A) Combine and mix the following components in a siliconized eppendorf tube: Step 4 (You may have to cut blindly due to low DNA concentrations).
H) Solubilize gels using QIAgen's gel extraction buffer at room temperature. Add 500uL QG buffer to each gel slice and shake on the vortexer for 30minutes. I) Purify DNA using a QIAquick PCR Purification Kit. Elute in 25ul EB.
Step 5: Amplification of adapter-modified DNA fragments and gel purification. Because we make libraries from both the small (200-400 bp) and the big (400-600 bp) size selected DNA fragments, we prepare 2 amplification reactions (and 2 libraries) per ChIP sample.
We also prepare a 200-400 bp and a 400-600 bp size-selected input library. Therefore, if all 6 histone modifications are analyzed, there will be 14 amplification reactions and 14 libraries ( E) Allow beads to dry. This may take anywhere from 10-20 minutes.
F) Add 30ul of EB buffer and shake on the vortexer for 30 minutes.
G) Place tubes back in magnetic stands to collect DNA; transfer liquid to siliconized Eppendorf tubes. Store libraries at -20ºC Step 7: Library Quantification and Confirmation To verify that a ChIP library has maintained a specific enrichment of target sites, perform qPCR on the ChIP-seq library using both positive targets and negative control primer pairs. The input library serves as a control to normalize the qPCR data to determine the relative enrichment of a given target.

Real-time quantitative PCR (qPCR)
Analyze the ChIP-seq sample as well as the appropriately sized input library for reference. Prepare a master reaction mix for each library with triplicate reactions per primer set. Add extra reagents for 10% of the total number of reagents to account for loss of volume. Add 15 µl of reaction mix to each PCR reaction well. Add 2 µl of primer mix to each well. Include a 70-95°C melting curve at the end of the qPCR program, reading all points or every 0.2°C.
Analyze library enrichments by qPCR as described above. Do not proceed with sequencing unless the positive targets are at least 20 fold enriched.