Loss of TGFBI, a secreted protein induced by transforming growth factor-β, has been implicated in cell proliferation, tumor progression, and angiogenesis by in vitro studies. However, in vivo antitumor functions of TGFBI as well as the underlying molecular mechanism are not well understood. To these aims, we have generated a mouse model with disruption of TGFBI genomic locus. Mice lacking TGFBI show a retarded growth and are prone to spontaneous tumors and 7,12-dimethylbenz(a)anthracene–induced skin tumors. In relation to wild-type (WT) mouse embryonic fibroblasts (MEF), TGFBI^−/− MEFs display increased frequencies of chromosomal aberration and micronuclei formation and exhibit an enhanced proliferation and early S-phase entry. Cyclin D1 is up-regulated in TGFBI^−/− MEFs, which correlates with aberrant activation of transcription factor cyclic AMP–responsive element binding protein (CREB) identified by chromatin immunoprecipitation and luciferase reporter assays. TGFBI reconstitution in TGFBI^−/− cells by either retroviral infection with WT TGFBI gene or supplement with recombinant mouse TGFBI protein in the culture medium leads to the suppression of CREB activation and cyclin D1 expression, and further inhibition of cell proliferation. Cyclin D1 up-regulation was also identified in most of the tumors arising from TGFBI^−/− mice. Our studies provide the first evidence that TGFBI functions as a tumor suppressor in vivo. [Cancer Res 2009;69(1):37–44]

C/EBPα is an essential transcription factor involved in regulating the expression or function of certain cell-cycle regulators, including in breast cancer cells. Although protein arginine methyltransferases have been shown to play oncogenic roles in a variety of cancers, little is known about the role of arginine methylation in regulating the antiproliferation activity of C/EBPα. Here, we report that the protein arginine methyltransferase 1 (PRMT1) is overexpressed in human breast cancer and that elevated PRMT1 correlates with cancer malignancy. RNA-sequencing analysis revealed that knockdown of PRMT1 in breast cancer cells is accompanied by a decrease in the expression of pro-proliferative genes, including cyclin D1. Furthermore, tandem affinity purification followed by mass spectrometry identified PRMT1 as a component of the C/EBPα complex. C/EBPα associated with and was methylated by PRMT1 at three arginine residues (R35, R156, and R165). PRMT1-dependent methylation of C/EBPα promoted the expression of cyclin D1 by blocking the interaction between C/EBPα and its corepressor HDAC3, which resulted in rapid growth of tumor cells during the pathogenesis of breast cancer. Inhibition of PRMT1 significantly impeded the growth of cancer cells from patients with triple-negative breast cancer. This evidence that PRMT1 mediates C/EBPα methylation sheds light on a novel pathway and potential therapeutic target in breast cancer. Significance: This study provides novel mechanistic insight of the role of the arginine methyltransferase PRMT1 in breast cancer pathogenesis.

Histone lysine (K) residues, which are modified by methyl- and acetyl-transferases, diversely regulate RNA synthesis. Unlike the ubiquitously activating effect of histone K acetylation, the effects of histone K methylation vary with the number of methyl groups added and with the position of these groups in the histone tails. Histone K demethylases (KDMs) counteract the activity of methyl-transferases and remove methyl group(s) from specific K residues in histones. KDM3A (also known as JHDM2A or JMJD1A) is an H3K9me2/1 demethylase. KDM3A performs diverse functions via the regulation of its associated genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanism by which the activity of KDM3A is regulated is largely unknown. Here, we demonstrated that mitogen- and stress-activated protein kinase 1 (MSK1) specifically phosphorylates KDM3A at Ser264 (p-KDM3A), which is enriched in the regulatory regions of gene loci in the human genome. p-KDM3A directly interacts with and is recruited by the transcription factor Stat1 to activate p-KDM3A target genes under heat shock conditions. The demethylation of H3K9me2 at the Stat1 binding site specifically depends on the co-expression of p-KDM3A in the heat-shocked cells. In contrast to heat shock, IFN-γ treatment does not phosphorylate KDM3A via MSK1, thereby abrogating its downstream effects. To our knowledge, this is the first evidence that a KDM can be modified via phosphorylation to determine its specific binding to target genes in response to thermal stress. Histone methylation regulates gene expression and can have drastic consequences for health if the process is defective. Histone lysine demethylases (KDMs) counteract the activity of methyl-transferases and remove methyl group(s) from histones. KDM3A is a H3K9me2/1 demethylase that performs diverse functions via the regulation of its target genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanisms underlying KDM3A regulation of specific genes at specific times are largely unknown. Here we found that a physiological stress—elevated temperature—induces KDM3A phosphorylation in human cells via the MSK1 kinase. This phosphorylated form of KDM3A directly interacts with the transcription factor Stat1, which enables Stat1 to recruit KDM3A to Stat1-binding sequences at the promoters of specific target genes. KDM3A then acts to demethylate H3K9me2/1 at these targets, thereby causing specific gene expression in response to the thermal stress. We conclude that heat shock can affect the expression of many genes in human cells via a novel activation mechanism that is centered around the phosphorylation of KDM3A.

Sox2 is a key component of the transcription factor network that maintains the pluripotent state of embryonic stem cells (ESCs). Sox2 is regulated by multiple post-translational modifications, including ubiquitination, sumoylation, acetylation and phosphorylation. Here we report that Sox2 is in association with and methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein arginine methyltransferase that plays a pivotal role in ESCs. We found that CARM1 facilitates Sox2-mediated transactivation and directly methylates Sox2 at arginine 113. This methylation event enhances Sox2 self-association. Furthermore, the physiological retention of Sox2 on chromatin restricts the Sox2 methylation level. Our study reveals the direct regulation of Sox2 by CARM1 that sheds lights on how arginine methylation signals are integrated into the pluripotent transcription factor network.

The regulation of gene expression via the histone code has, for the most part, revealed that histone modifications cause the recruitment of adaptor proteins that indirectly regulate the synthesis of RNA. Using purified factors to assemble and modify the chromatin and to transcribe the DNA, we investigated whether modifications of histones may directly impact the RNA polymerase II transcription process. We screened proteins known to modify histones for effects on transcription, and we found that the mitogen- and stress-induced kinase, MSK1, inhibited RNA synthesis. Inhibition of transcription by MSK1 was most sensitive when the template was in chromatin, as naked DNA templates were resistant to the effects of MSK1. We found that MSK1 phosphorylated histone H2A on serine 1, and mutation of serine 1 to alanine blocked the inhibition of transcription by MSK1. Furthermore, we found that acetylation of histone H3 by the p300 and CREB-binding protein associated factor, PCAF, suppressed the kinase-dependent inhibition of transcription. These results suggest that acetylation of histones may stimulate transcription by suppressing an inhibitory phosphorylation by a kinase as MSK1.