Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation.

The CCCTC-binding factor (CTCF) works together with the cohesin complex to drive the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionately affect genes that are bound by CTCF at the promoter and are dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which were restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with the transcription of over 2,000 genes across a diverse set of adult tissues. Taken together, the results of our study show that CTCF binding to promoters may promote long-distance enhancer-dependent transcription at specific genes in diverse cell types.

NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes.

Our understanding of transcription by RNA polymerase II (Pol II) is limited by our knowledge of the factors that mediate this critically important process. Here we describe the identification of NDF, a nucleosome-destabilizing factor that facilitates Pol II transcription in chromatin. NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by Pol II through nucleosomes in a purified and defined transcription system as well as in cell nuclei. Upon transcriptional induction, NDF is recruited to the transcribed regions of thousands of genes and colocalizes with a subset of H3K36me3-enriched regions. Notably, the recruitment of NDF to gene bodies is accompanied by an increase in the transcript levels of many of the NDF-enriched genes. In addition, the global loss of NDF results in a decrease in the RNA levels of many genes. In humans, NDF is present at high levels in all tested tissue types, is essential in stem cells, and is frequently overexpressed in breast cancer. These findings indicate that NDF is a nucleosome-destabilizing factor that is recruited to gene bodies during transcriptional activation and facilitates Pol II transcription through nucleosomes.

MPE-seq, a new method for the genome-wide analysis of chromatin structure.

The analysis of chromatin structure is essential for the understanding of transcriptional regulation in eukaryotes. Here we describe methidiumpropyl-EDTA sequencing (MPE-seq), a method for the genome-wide characterization of chromatin that involves the digestion of nuclei withMPE-Fe(II) followed by massively parallel sequencing. Like micrococcal nuclease (MNase), MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes. However, there are differences in the cleavage of nuclear chromatin by MPE-Fe(II) relative to MNase. Most notably, immediately upstream of the transcription start site of active promoters, we frequently observed nucleosome-sized (141-190 bp) and subnucleosome-sized (such as 101-140 bp) peaks of digested chromatin fragments with MPE-seq but not with MNase-seq. These peaks also correlate with the presence of core histones and could thus be due, at least in part, to noncanonical chromatin structures such as labile nucleosome-like particles that have been observed in other contexts. The subnucleosome-sized MPE-seq peaks exhibit a particularly distinct association with active promoters. In addition, unlike MNase, MPE-Fe(II) cleaves nuclear DNA with little sequence bias. In this regard, we found that DNA sequences at RNA splice sites are hypersensitive to digestion by MNase but not by MPE-Fe(II). This phenomenon may have affected the analysis of nucleosome occupancy over exons. These findings collectively indicate that MPE-seq provides a unique and straightforward means for the genome-wide analysis of chromatin structure with minimal DNA sequence bias. In particular, the combined use of MPE-seq and MNase-seq enables the identification of noncanonical chromatin structures that are likely to be important for the regulation of gene expression.

Prenucleosomes and Active Chromatin.

Chromatin consists of nucleosomes as well as nonnucleosomal histone-containing particles. Here we describe the prenucleosome, which is a stable conformational isomer of the nucleosome that associates with ∼80 bp DNA. Prenucleosomes are formed rapidly upon the deposition of histones onto DNA and can be converted into canonical nucleosomes by an ATP-driven chromatin assembly factor such as ACF. Different lines of evidence reveal that there are prenucleosome-sized DNA-containing particles with histones in the upstream region of active promoters. Moreover, p300 acetylates histone H3K56 in prenucleosomes but not in nucleosomes, and H3K56 acetylation is found at active promoters and enhancers. These findings therefore suggest that there may be prenucleosomes or prenucleosome-like particles in the upstream region of active promoters. More generally, we postulate that prenucleosomes or prenucleosome-like particles are present at dynamic chromatin, whereas canonical nucleosomes are at static chromatin.

CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells.

The pluripotency of embryonic stem cells (ESCs) is maintained by a small group of master transcription factors including Oct4, Sox2 and Nanog. These core factors form a regulatory circuit controlling the transcription of a number of pluripotency factors including themselves. Although previous studies have identified transcriptional regulators of this core network, the cis-regulatory DNA sequences required for the transcription of these key pluripotency factors remain to be defined. We analyzed epigenomic data within the 1.5 Mb gene-desert regions around the Sox2 gene and identified a 13kb-long super-enhancer (SE) located 100kb downstream of Sox2 in mouse ESCs. This SE is occupied by Oct4, Sox2, Nanog, and the mediator complex, and physically interacts with the Sox2 locus via DNA looping. Using a simple and highly efficient double-CRISPR genome editing strategy we deleted the entire 13-kb SE and characterized transcriptional defects in the resulting monoallelic and biallelic deletion clones with RNA-seq. We showed that the SE is responsible for over 90% of Sox2 expression, and Sox2 is the only target gene along the chromosome. Our results support the functional significance of a SE in maintaining the pluripotency transcription program in mouse ESCs.

Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark.

We report the identification of a new type of histone mark, lysine 2-hydroxyisobutyrylation (Khib), and identify the mark at 63 human and mouse histone Khib sites, including 27 unique lysine sites that are not known to be modified by lysine acetylation (Kac) and lysine crotonylation (Kcr). This histone mark was initially identified by MS and then validated by chemical and biochemical methods. Histone Khib shows distinct genomic distributions from histone Kac or histone Kcr during male germ cell differentiation. Using chromatin immunoprecipitation sequencing, gene expression analysis and immunodetection, we show that in male germ cells, H4K8hib is associated with active gene transcription in meiotic and post-meiotic cells. In addition, H4K8ac-associated genes are included in and constitute only a subfraction of H4K8hib-labeled genes. The histone Khib mark is conserved and widely distributed, has high stoichiometry and induces a large structural change. These findings suggest its critical role on the regulation of chromatin functions.

Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF.

Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here, we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 and the ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable nonnucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF. The histone-DNA intermediate does not supercoil DNA like a canonical nucleosome, but has a nucleosome-like appearance by atomic force microscopy. This intermediate contains all four core histones, lacks NAP1, and is formed by the initial deposition of histones H3-H4. Conversion of the intermediate into histone H1-containing chromatin results in increased resistance to micrococcal nuclease digestion. These findings suggest that the histone-DNA intermediate corresponds to nascent nucleosome-like structures, such as those observed at DNA replication forks. Related complexes might be formed during other chromatin-directed processes such as transcription, DNA repair, and histone exchange.

A cis-regulatory map of the Drosophila genome.

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

Mi2beta shows chromatin enzyme specificity by erasing a DNase I-hypersensitive site established by ACF.

ATP-dependent chromatin-remodeling enzymes are linked to changes in gene expression; however, it is not clear how the multiple remodeling enzymes found in eukaryotes differ in function and work together. In this report, we demonstrate that the ATP-dependent remodeling enzymes ACF and Mi2beta can direct consecutive, opposing chromatin-remodeling events, when recruited to chromatin by different transcription factors. In a cell-free system based on the immunoglobulin heavy chain gene enhancer, we show that TFE3 induces a DNase I-hypersensitive site in an ATP-dependent reaction that requires ACF following transcription factor binding to chromatin. In a second step, PU.1 directs Mi2beta to erase an established DNase I-hypersensitive site, in an ATP-dependent reaction subsequent to PU.1 binding to chromatin, whereas ACF will not support erasure. Erasure occurred without displacing the transcription factor that initiated the site. Other tested enzymes were unable to erase the DNase I-hypersensitive site. Establishing and erasing the DNase I-hypersensitive site required transcriptional activation domains from TFE3 and PU.1, respectively. Together, these results provide important new mechanistic insight into the combinatorial control of chromatin structure.

Activation of 12/23-RSS-dependent RAG cleavage by hSWI/SNF complex in the absence of transcription.

Maintenance of genomic integrity during antigen receptor gene rearrangements requires (1) regulated access of the V(D)J recombinase to specific loci and (2) generation of double-strand DNA breaks only after recognition of a pair of matched recombination signal sequences (RSSs). Here we recapitulate both key aspects of regulated recombinase accessibility in a cell-free system using plasmid substrates assembled into chromatin. We show that recruitment of the SWI/SNF chromatin-remodeling complex to both RSSs increases coupled cleavage by RAG1 and RAG2 proteins. SWI/SNF functions by altering local chromatin structure in the absence of RNA polymerase II-dependent transcription or histone modifications. These observations demonstrate a direct role for cis-sequence-regulated local chromatin remodeling in RAG1/2-dependent initiation of V(D)J recombination.

Histone H4-K16 acetylation controls chromatin structure and protein interactions.

Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.

gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair.

Phosphorylated histone H2AX (gamma-H2AX) forms foci over large chromatin domains surrounding double-stranded DNA breaks (DSB). These foci recruit DSB repair proteins and dissolve during or after repair is completed. How gamma-H2AX is removed from chromatin remains unknown. Here, we show that protein phosphatase 2A (PP2A) is involved in removing gamma-H2AX foci. The PP2A catalytic subunit [PP2A(C)] and gamma-H2AX coimmunoprecipitate and colocalize in DNA damage foci and PP2A dephosphorylates gamma-H2AX in vitro. The recruitment of PP2A(C) to DNA damage foci is H2AX dependent. When PP2A(C) is inhibited or silenced by RNA interference, gamma-H2AX foci persist, DNA repair is inefficient, and cells are hypersensitive to DNA damage. The effect of PP2A on gamma-H2AX levels is independent of ATM, ATR, or DNA-PK activity.

Enhancement of pleural dissemination and lymph node metastasis of intrathoracic lung cancer cells by vascular endothelial growth factors (VEGFs).

The expression of vascular endothelial growth factors (VEGFs) in tumors including lung cancer is considered to be associated with tumor development via capillary and lymph vessel neogenesis. Dissemination of the tumor cells to the pleura or regional lymph nodes is a critical poor prognostic factor for lung cancer patients. To investigate how VEGFs expressed in the intrathoracic infiltrating lung cancer cells participate in disease progression, we established stably VEGF-A-, VEGF-C-, VEGF-D-, VEGF-A and VEGF-C-, and VEGF-A and VEGF-D-expressing large cell lung cancer clones (TKB5/VEGF-A, TKB5/VEGF-C, TKB5/VEGF-D, TKB5/VEGF-A/C, and TKB5/VEGF-A/D), orthotopically inoculated these into the right thoracic cavity (i.t.) of nude mice, and evaluated the subsequent development of lung lesion, pleural effusion, pleural dissemination, and lymph node metastasis. While there were no significant differences either in culture or in subcutaneous tumor cell growth between the empty vector-transfected group (TKB5/empty) and each transfectant, the i.t. model demonstrated significantly different biological properties between the transfectants. TKB5/empty-inoculated mice frequently developed a large tumor on the pleura without pleural effusion, dissemination, or lymph node (LN) metastasis. In contrast, VEGF-A promoted a bloody pleural effusion (6/14), and VEGF-A and VEGF-D frequently generated pleural dissemination (11/14 and 9/11, respectively). Although both VEGF-C and VEGF-D generated LN metastasis (6/10 and 8/11, respectively), the locations of the metastasized LNs were quite different. TKB5/VEGF-C metastasized on the same side of axillary LNs as i.t. (right axillary LNs), whereas TKB5/VEGF-D metastasized to the mediastinal and left axillary and/or cervical LNs. Since the TKB5/VEGF-A/C or TKB5/VEGF-A/D co-transfectants revealed overlapping tumor progression patterns of VEGF-A and VEGF-C or VEGF-D, the metastatic LNs had abundant new capillaries and were larger than those of TKB5/VEGF-C or TKB5/VEGF-D-inoculated mice. Our results clearly demonstrate that VEGF-A secreted from intrathoracic lung cancer cells plays important roles in producing pleural effusion, dissemination, and capillary neogenesis, that VEGF-C is involved in LN metastasis, and VEGF-D in pleural dissemination and LN metastasis. It is most likely, however, that the mechanisms by which VEGF-C promotes LN metastasis are different from those of VEGF-D. The regulation of the expression of VEGFs in intrathoracic lung cancer cells might be a useful therapeutic approach to inhibiting tumor development and improving patient prognosis.

Combinatorial control of DNase I-hypersensitive site formation and erasure by immunoglobulin heavy chain enhancer-binding proteins.

DNase I-hypersensitive sites in cellular chromatin are usually believed to be nucleosome-free regions generated by transcription factor binding. Using a cell-free system we show that hypersensitivity does not simply correlate with the number of DNA-bound proteins. Specifically, the leucine zipper containing basic helix-loop-helix protein TFE3 was sufficient to induce a DNase I-hypersensitive site at the immunoglobulin heavy chain micro enhancer in vitro. TFE3 enhanced binding of an ETS protein PU.1 to the enhancer. However, PU.1 binding erased the DNase I-hypersensitive site without abolishing TFE3 binding. Furthermore, TFE3 binding enhanced transcription in the presence and absence of a hypersensitive site, whereas endonuclease accessibility correlated strictly with DNase I hypersensitivity. We infer that chromatin constraints for transcription and nuclease sensitivity can differ.

Colliding primary lung cancers of adenosquamous carcinoma and large cell neuroendocrine carcinoma.

We report an extremely rare case of primary lung cancer showing various histological elements diagnosed as the collision of an adenosquamous carcinoma and a large cell neuroendocrine carcinoma by loss of heterozygosity (LOH) analysis of the human androgen receptor (AR) and phosphoglycerate kinase (PGK-1) genes. The tumor exhibited a tiny ground-glass opaque shadow suggesting atypical adenomatous hyperplasia 18 months prior to surgery. However, the tumor grew rapidly, and the resected tumor consisted of two closely located nodules. The larger nodule was composed of well-differentiated adenocarcinomatous and moderately to poorly differentiated squamous cell carcinomatous elements, while the smaller nodule consisted of a large cell neuroendocrine carcinomatous element with partial squamoid differentiation having focal continuity with the adenocarcinomatous element. Both the adenocarcinomatous and squamous cell carcinomatous elements revealed transitional features and LOH of AR and PGK-1 genes, while the large cell neuroendocrine carcinomatous element showed a monoclonal pattern but possessed both alleles of AR and PGK-1 genes. From these clinical and pathological results, the parental cell of the large cell neuroendocrine carcinomatous element was considered to be different from that of the adenosquamous carcinomatous element.

Hepatoid adenocarcinoma in the lung.

We have experienced a very rare case of hepatoid adenocarcinoma in the lung. A 55-year-old male with a history of smoking was diagnosed as adenocarcinoma of the right S2, and underwent resection of the right upper lobe and dissection of the hilum and mediastinal lymph nodes (complete resection). Pathological examination revealed cuboid atypical cells arranged in a papillary or trabecular fashion, and a proliferating pattern in most part of the tumor resembling that of hepatocellular carcinoma. Immunohistochemistry study showed alpha-fetoprotein (AFP)-positive tumor cells, hence the diagnosis of hepatoid adenocarcinoma was confirmed. Postoperative pathological staging classified the patient as having as p-T2NOMO, p-stage I B disease. The serum AFP level was not determined before surgery, but the value decreased gradually after surgery and has since remained within the normal range. At present, 32 months after the operation, no signs of recurrence have been observed. Hepatoid adenocarcinoma in the lung has been reported in eight patients with the present one being the ninth case. All were males, the tumor diameter was large and the patient prognoses were mostly poor, but at early stage two cases of long-term survival have been reported indicating that the stage is the most important prognostic factor also for rare tumor.