17-611 Sigma-AldrichMagna ChIP™ G - Chromatin Immunoprecipitation Kit

We have previously demonstrated that clock genes contribute to the homeostatic aspect of sleep regulation. Indeed, mutations in some clock genes modify the markers of sleep homeostasis and an increase in homeostatic sleep drive alters clock gene expression in the forebrain. Here, we investigate a possible mechanism by which sleep deprivation (SD) could alter clock gene expression by quantifying DNA-binding of the core-clock transcription factors CLOCK, NPAS2, and BMAL1 to the cis-regulatory sequences of target clock genes in mice. Using chromatin immunoprecipitation (ChIP), we first showed that, as reported for the liver, DNA-binding of CLOCK and BMAL1 to target clock genes changes in function of time-of-day in the cerebral cortex. Tissue extracts were collected at ZT0 (light onset), -6, -12, and -18, and DNA enrichment of E-box or E'-box containing sequences was measured by qPCR. CLOCK and BMAL1 binding to Cry1, Dbp, Per1, and Per2 depended on time-of-day, with maximum values reached at around ZT6. We then observed that SD, performed between ZT0 and -6, significantly decreased DNA-binding of CLOCK and BMAL1 to Dbp, consistent with the observed decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was also decreased by SD, although SD is known to increase Per2 expression in the cortex. DNA-binding to Per1 and Cry1 was not affected by SD. Our results show that the sleep-wake history can affect the clock molecular machinery directly at the level of chromatin binding thereby altering the cortical expression of Dbp and Per2 and likely other targets. Although the precise dynamics of the relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive, the results also suggest that part of the reported circadian changes in DNA-binding of core clock components in tissues peripheral to the suprachiasmatic nuclei could, in fact, be sleep-wake driven.

Nonrandom cytogenetic abnormalities of chromosomes 6, 7, and 17 have been reported within low-grade endometrial stromal sarcomas (LGESSs), and among these abnormalities, the t(7;17)(p15;q21) is the most common aberration described. Previously we had shown that this translocation joins 2 genes, JAZF1 and JJAZ1, located on chromosomes 7 and 17, respectively. To determine the frequency of the t(7;17), we analyzed 4 stromal nodules and 24 LGESS by both reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization (FISH). In addition, we examined 4 cases of highly cellular leiomyoma, a benign morphologic mimic of LGESS. Overall, evidence for the JAZF1-JJAZ1 fusion was found in 60% of endometrial stromal neoplasms analyzed (8/16 ESS and 4/4 stromal nodules). One LGESS demonstrated only rearrangement of 7p15 by FISH analysis and karyotypic analysis of this case showed t(6;7)(p21;p15). The fusion was not detected in any highly cellular leiomyomas. Our data suggest that the JAZF1-JJAZ1 fusion is a frequent, although nonuniform, feature of endometrial stromal neoplasia, irrespective of benign versus malignant classification and smooth muscle differentiation. In addition, the detection of the fusion by reverse transcriptase-polymerase chain reaction or FISH for JJAZ1 at 7p15 may be diagnostically useful.

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

SUZ12 is a Polycomb group protein that forms Polycomb repressive complexes (PRC2/3) together with EED and histone methyltransferase EZH2. Although the essential role of SUZ12 in regulating the activity of the PRC2/3 complexes has been demonstrated, additional function of this protein was suggested. Here, we show that SUZ12 interacts with WD-repeat protein MEP50 in vitro and in vivo. We show that the MEP50 binds histone H2A selectively among core histones, and mediates transcriptional repression of protein arginine methyltransferase PRMT5, which is known to methylate H2A and H4. These results suggest that SUZ12 might have a role in transcriptional regulation through physical interaction with MEP50 that can be an adaptor between PRMT5 and its substrate H2A.

Chromatin immunoprecipitation (ChIP) is a well-established procedure to investigate interactions between proteins and DNA. Coupled with whole-genome DNA microarrays, ChIPS allow one to determine the entire spectrum of in vivo DNA binding sites for any given protein. The design and analysis of ChIP-microarray (also called ChIP-chip) experiments differ significantly from the conventions used for locus ChIP approaches and ChIP-chip experiments, and these differences require new methods of analysis. In this light, we review the design of DNA microarrays, the selection of controls, the level of repetition required, and other critical parameters for success in the design and analysis of ChIP-chip experiments, especially those conducted in the context of mammalian or other relatively large genomes.

Endometrial stromal tumors (ESTs), including low-grade endometrial stromal sarcomas (LGESSs) and endometrial stromal nodules (ESNs) of classic histology, exhibit characteristic morphologic features and contain the nonrandom t(7;17)(p15; q21), which results in the fusion of two novel genes, JAZF1 and JJAZ1. ESTs may pose diagnostic challenges when they involve extrauterine sites, present as metastases, or display variant histologic appearances. The aim of this study was to evaluate the frequency of the JAZF1-JJAZ1 gene fusion among primary uterine, metastatic, and primary extrauterine ESTs of various histologic types and its role as a possible diagnostic adjunct. Using a nonnested reverse transcriptase-polymerase chain reaction approach, we assayed for JAZF1-JJAZ1 gene fusion transcripts in 10 cases with available fresh-frozen tissue. These included five primary uterine (two classic, one mixed smooth muscle, and one epithelioid LGESS; one classic ESN), four metastatic (two fibromyxoid, one classic, and one epithelioid LGESS), and one extrauterine (classic LGESS) tumor. The same primer set and assay conditions were used on five additional paraffin-embedded cases with adequate RNA, including three primary uterine (one fibromyxoid and one mixed smooth muscle LGESS; 1 mixed smooth muscle ESN) and two intraabdominal recurrent (two mixed smooth muscle LGESSs) ESTs. Two cellular leiomyomas and one ESS cell line (ESS-1) without the t(7;17) at the cytogenetic level were run in parallel as controls. JAZF1-JJAZ1 gene fusion transcripts were detected in five (33%) of 15 ESTs, including three of eight primary uterine, one of four metastatic, one of one extrauterine, and none of two recurrent cases. Most ESTs of classic histology showed evidence of JAZF1-JJAZ1 fusion (4 of 5 cases), whereas only one mixed smooth muscle ESN of 10 variant cases was positive. Positivity for JAZF1-JJAZ1 fusion transcripts was found in four of 10 fresh-frozen samples and in one of five paraffin-embedded ESTs. The control specimens were all negative. In conclusion, our data suggest that ESTs are genetically heterogeneous, with the prevalence of the JAZF1-JJAZ1 fusion being highest among ESTs of classic histology. Hence, the diagnostic utility of a JAZF1-JJAZ1 fusion transcript assay in ESTs may be limited to the classic histologic subset.