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Idiopathic pulmonary fibrosis (IPF) is a chronic human disease with persistent destruction of lung parenchyma. Transforming growth factor-β1 (TGF-β1) signaling plays a pivotal role in the initiation and pathogenesis of IPF. As shown herein, TGF-β1 signaling down-regulated not only peroxisome biogenesis but also the metabolism of these organelles in human IPF fibroblasts. In vitrocell culture observations in human fibroblasts and human lung tissue indicated that peroxisomal biogenesis and metabolic proteins were significantly down-regulated in the lung of 1-month–old transgenic mice expressing a constitutively active TGF-β type I receptor kinase (ALK5). The peroxisome biogenesis protein peroxisomal membrane protein Pex13p (PEX13p) as well as the peroxisomal lipid metabolic enzyme peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and antioxidative enzyme catalase were highly up-regulated in TGF-β type II receptor and Smad3 knockout mice. This study reports a novel mechanism of peroxisome biogenesis and metabolic regulation via TGF-β1–Smad signaling: interaction of the Smad3 transcription factor with the PEX13gene in chromatin immunoprecipitation–on–chip assay as well as in a bleomycin-induced pulmonary fibrosis model applied to TGF-β type II receptor knockout mice. Taken together, data from this study suggest that TGF-β1 participates in regulation of peroxisomal biogenesis and metabolism via Smad-dependent signaling, opening up novel strategies for the development of therapeutic approaches to inhibit progression of pulmonary fibrosis patients with IPF.

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8–ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.

Protein arginine methyltransferase 6 (PRMT6) catalyzes asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a). This mark has been reported to associate with silent genes. Here, we use a cell model of neural differentiation, which upon PRMT6 knockout exhibits proliferation and differentiation defects. Strikingly, we detect PRMT6-dependent H3R2me2a at active genes, both at promoter and enhancer sites. Loss of H3R2me2a from promoter sites leads to enhanced KMT2A binding and H3K4me3 deposition together with increased target gene transcription, supporting a repressive nature of H3R2me2a. At enhancers, H3R2me2a peaks co-localize with the active enhancer marks H3K4me1 and H3K27ac. Here, loss of H3R2me2a results in reduced KMT2D binding and H3K4me1/H3K27ac deposition together with decreased transcription of associated genes, indicating that H3R2me2a also exerts activation functions. Our work suggests that PRMT6 via H3R2me2a interferes with the deposition of adjacent histone marks and modulates the activity of important differentiation-associated genes by opposing transcriptional effects.

The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by chromatin immunoprecipitation sequencing (ChIP-seq) experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin 1 (IL-1)-induced H3K27ac and p65 binding. Inhibition of TAK1 or IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXCchemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50, and of AP-1 transcription factors to both promoters and enhancers. This study provides a high-resolution view of epigenetic changes occurring during the IL-1 response and allows the genome-wide identification of a distinct class of inducible p65 NF-κB-dependent enhancers in epithelial cells.

Insulators are multiprotein–DNA complexes that regulate the nuclear architecture. The DrosophilaCP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCPinsulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.

Sperm chromatin reveals two characteristic features in that protamines are the predominant nuclear proteins and remaining histones are highly acetylated. Histone H4 acetylated at lysine 12 (H4K12ac) is localized in the post-acrosomal region, while protamine-1 is present within the whole nucleus. Chromatin immunoprecipitation in combination with promoter array analysis allowed genome-wide identification of H4K12ac binding sites. Previously, we reported enrichment of H4K12ac at CTCF binding sites and promoters of genes involved in developmental processes. Here, we demonstrate that H4K12ac is enriched predominantly between ± 2 kb from the transcription start site. In addition, we identified developmentally relevant H4K12ac-associated promoters with high expression levels of their transcripts stored in mature sperm. The highest expressed mRNA codes for testis-specific PHD finger protein-7 (PHF7), suggesting an activating role of H4K12ac in the regulatory elements of this gene. H4K12ac-associated genes revealed a weak correlation with genes expressed at 4-cell stage human embryos, while 23 H4K12ac-associated genes were activated in 8-cell embryo and 39 in the blastocyst. Genes activated in 4-cell embryos are involved in gene expression, histone fold and DNA-dependent transcription, while genes expressed in the blastocyst were classified as involved in developmental processes. Immunofluorescence staining detected H4K12ac from the murine male pronucleus to early stages of embryogenesis. Aberrant histone acetylation within developmentally important gene promoters in infertile men may reflect insufficient sperm chromatin compaction, which may result in inappropriate transfer of epigenetic information to the oocyte.

Much of spermatid differentiation takes place in the absence of active transcription, but in the early phase, large amounts of mRNA are synthesized, translationally repressed, and stored. Most nucleosomal histones are then degraded, and chromatin is repackaged by protamines. For both transcription and the histone-to-protamine transition in differentiating spermatids, chromatin must be opened. This raises the question of whether two different processes exist. It is conceivable that for initiation of the histone-to-protamine transition, the already accessible, actively transcribed chromatin regions are utilized or vice versa. We analyzed the enrichment of different canonical TATA-box-binding, protein-associated factors and their variants in murine spermatids, diverse bromodomain-containing proteins, and components of the Polycomb repressive complexes PRC1 and PRC2 using quantitative PCR. We compared the enrichment of corresponding proteins in human and murine spermatids and analyzed the time frame of postmeiotic transcription and expression of histones, transition proteins, and protamines in human and murine spermatids using immunohistology. We correlated the expression of different transcription factors and bromodomain-containing proteins and the pattern of acetylated histones to active transcription and to the histone-to-protamine transition in both human and murine spermatids. Our findings suggest that differentiating spermatids use both common and specific features to open chromatin first for transcription and subsequently for histone-to-protamine transition.