H3Y41 ChiP-Seq in K1106
Cooperative Epigenetic Modulation by Cancer Amplicon Genes
ChIP-enriched DNA were used to create adapter-ligated libraries for massively  parallel sequencing with the ChIP-Seq DNA Sample Prep Kit (Illumina) following  manufacturer’s protocol with the following modifications: adapters were diluted 1:20;  200bp-600bp fragment size range was selected with agarose gel electrophoresis; libraries  were PCR-enriched for 21 cycles with 45” extension steps. Standard Solexa/Illumina kits,  reagents, protocols and instruments were used for generating clusters on flowcells and  sequencing the 2 lanes of each library on a Genome Analyzer GAII (Illumina). Images  from Illumina sequencer GAII were processed with Illumina GOAT pipeline for final  base calling and quality score at each base. Resulting sequences were then aligned to  human genome build 36 (hg18) with ELAND_EXTENDED software. Reads with 36bp  in length from same library in multiple lanes of same or different flowcell were pooled by  concatenating their alignment files before subjected to summarization of read alignment.  Only reads with up to two miss matches but uniquely mapping to reference genome were  counted.  In analyzing the H3Y41p Chip-seq data, only unique tags were counted within a  given experiment, so that if multiple copies of identical tag sequences were found, they  were analyzed as though there was only a single instance. A total of 1,875,208 unique  tags were identified in the control experiment and 1,179,331 unique tags were identified  in the test experiment, for a total of 3,054,539 total tags. The tags were collected into bins  of 25bp and then extended an additional 350 bp from their origin. To identify peaks, we  counted the number of total extended tags that intersected each bin, and identified peaks  made up of consecutive bins each of which were intersected by at least 7 of the extended  tags. The value 7 was chosen so as to optimize the proportion of peaks that were within  2kb of a gene.
Chromosome band 9p24 is frequently amplified in primary mediastinal B-cell lymphoma  (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we  screened an RNA interference library targeting amplicon genes and thereby identified  JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas.  Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing  tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3,  promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone  phosphorylation, was silenced following JAK2 and JMJD2C inhibition, with a  corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively  remodel the PMBL and HL epigenome, offering a mechanistic rationale for the  development of JAK2 and JMJD2C inhibitors in these diseases.