Differential glucocorticoid receptor-mediated transcription mechanisms.

Nuclear receptors such as the glucocorticoid receptor (GR) are ligand-dependent transcription factors that mediate transcription of target genes by recruiting factors that modulate chromatin structure. In this study, curcumin, a compound known to inhibit GR-mediated transcription, was used to examine the different mechanisms by which GR regulates transcription. The mechanisms of transcription regulation of metallothioneine-2A (MT2A) and solute carrier family 19 member 2 (SLC19A2), two GR target genes where the hormone-dependent gene activation is inhibited or unaffected by curcumin treatment, respectively, were analyzed by chromatin immunoprecipitation and RT-PCR experiments. The data suggest that the loss of hormone-dependent MT2A gene expression is due to the inhibition of continued transcription activity after initial assembly of the transcription machinery. In contrast, the hormone-dependent SLC19A2 gene expression is maintained because the continued transcription output after assembly of transcription machinery is unaffected by curcumin. These results suggest that the two GR target genes use alternate mechanisms to regulate expression levels at the level of continued transcription output after transcription machinery assembly.

Modulating molecular chaperone Hsp90 functions through reversible acetylation.

The molecular chaperone protein Hsp90 is a key regulator of approximately 100 'client' proteins crucial for numerous cell signaling processes. Consequently, understanding the molecular underpinnings that regulate Hsp90 activity is an important biological endeavor. Exciting new results now suggest that, at least for nuclear receptor activity, Hsp90 function is directly regulated by histone deacetylase 6 (HDAC6). These observations have consequences for various biological processes and potentially important implications for the development of cancer therapeutics.

Dynamics of coactivator recruitment and chromatin modifications during nuclear receptor mediated transcription.

The mechanisms and interplay of coactivators that underlie transcription activation is a critical avenue of investigation in biology today. Using nuclear receptor (NR) mediated transcription activation as a model, the nature of coactivator recruitment and chromatin modifications has been found to be highly dynamic. Progress in understanding the kinetics and regulation of coactivator recruitment, and subsequent effects on transcriptional readout, has greatly improved our understanding of nuclear receptor mediated transcription, the subject of discussion in this 'At the Cutting Edge' review.

Dynamic histone acetylation/deacetylation with progesterone receptor-mediated transcription.

Histone acetylation is a highly dynamic posttranslational modification that plays an important role in gene expression. Previous work showed that promoter histone deacetylation is accompanied by progesterone receptor (PR)-mediated activation of the mouse mammary tumor virus (MMTV) promoter. We investigated the role of this deacetylation and found that this histone deacetylation is not a singular event. In fact, histone acetylation at the MMTV promoter is highly dynamic, with an initial increase in acetylation followed by an eventual net deacetylation of histone H4. The timing of increase in acetylation of H4 coincides with the time at which PR, RNA polymerase II, and histone acetyltransferases cAMP response element-binding protein (CREB)-binding protein and p300 are recruited to the MMTV promoter. The timing in which histone H4 deacetylation occurs (after PR and RNA polymerase II recruitment) and the limited effect that trichostatin A and small interfering RNA knockdown of histone deacetylase (HDAC)3 have on MMTV transcription suggests that this deacetylation activity is not required for the initiation of PR-mediated transcription. Interestingly, two HDACs, HDAC1 and HDAC3, are already present at the MMTV before transcription activation. HDAC association at the MMTV promoter fluctuates during the hormone treatment. In particular, HDAC3 is temporarily undetected at the MMTV promoter within minutes after hormone treatment when the histone H4 acetylation increases but returns to the promoter near the time when histone acetylation levels start to decline. These results demonstrate the dynamic nature of coactivator/corepressor-promoter association and histone modifications such as acetylation during a transcription activation event.

CARM1 methylates MED12 to regulate its RNA-binding ability.

The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain-containing effector molecule, TDRD3. Chromatin immunoprecipitation-seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.

hSWI/SNF-catalyzed nucleosome sliding does not occur solely via a twist-diffusion mechanism.

Nucleosome remodeling by the hSWI/SNF complex and other chromatin remodeling complexes can cause translocation (sliding) of the histone octamer in cis along DNA. Structural and biochemical evidence suggest that sliding involves a DNA twist-diffusion process whereby the DNA rotates about the helical axis without major displacement from the surface of the nucleosome and that this process may be driven by torsional stress within the DNA. We report that hSWI/SNF efficiently catalyzes sliding of nucleosomes containing branched DNAs as steric blocks to twist-diffusion and a nick to allow dissipation of torsional stress within the nucleosome. These results suggest that SWI/SNF-catalyzed nucleosome sliding does not occur exclusively via a simple twist-diffusion mechanism and support models in which the DNA maintains its rotational orientation to and is at least partially separated from the histone surface during nucleosome translocation.

Nucleosome remodeling by the human SWI/SNF complex requires transient global disruption of histone-DNA interactions.

We utilized a site-specific cross-linking technique to investigate the mechanism of nucleosome remodeling by hSWI/SNF. We found that a single cross-link between H2B and DNA virtually eliminates the accumulation of stably remodeled species as measured by restriction enzyme accessibility assays. However, cross-linking the histone octamer to nucleosomal DNA does not inhibit remodeling as monitored by DNase I digestion assays. Importantly, we found that the restriction enzyme-accessible species can be efficiently cross-linked after remodeling and that the accessible state does not require continued ATP hydrolysis. These results imply that the generation of stable remodeled states requires at least transient disruption of histone-DNA interactions throughout the nucleosome, while hSWI/SNF-catalyzed disruption of just local histone-DNA interactions yields less-stable remodeled states that still display an altered DNase I cleavage pattern. The implications of these results for models of the mechanism of SWI/SNF-catalyzed nucleosome remodeling are discussed.

Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.

Nicotinamide uncouples hormone-dependent chromatin remodeling from transcription complex assembly.

Sirtuins, homologs of the yeast SIR2 family, are protein deacetylases that require nicotinamide adenosine dinucleotide as cofactor. To determine whether the sirtuin family of deacetylases is involved in progesterone receptor (PR)-mediated transcription, the effect of sirtuin inhibitor, nicotinamide (NAM), was monitored in T47D breast cancer cells. NAM suppressed hormone-dependent activation of PR-regulated genes in a dose-dependent manner. Surprisingly, NAM-mediated inhibition of PR-mediated transcription occurs independently of SIRT1 and PARP1. Chromatin immunoprecipitation experiments did not show that PR binding nor that of the coactivators CBP and SRC3 was compromised. Consistent with the recruitment of the BRG1 chromatin remodeling complex, promoter chromatin remodeling still occurs despite NAM inhibition of PR transactivation. Rather, we show that this inhibition of transcription is due to dramatic loss of recruitment of the basal transcriptional machinery to the promoter. These results show that NAM uncouples promoter chromatin remodeling from transcription preinitiation complex assembly and suggest the existence of vital NAM-regulated steps required for promoter chromatin remodeling and basal transcription complex communication.

ATP-dependent chromatin remodeling complexes and their role in nuclear receptor-dependent transcription in vivo.

Nuclear receptors (NRs) are ligand-dependent transcription factors that mediate transcription of target genes in chromatin. Modulation of chromatin structure plays an important part in the NR-mediated transcription process. ATP-dependent chromatin remodeling complexes have been shown to be intimately involved in NR-mediated transcription. In this review, we examine the role of chromatin remodeling complexes in facilitating the recruitment of coregulators and basal transcription factors. In addition, the role of subunit specificity within the chromatin remodeling complexes, the complexes' influence on remodeling activity, and complexes' recruitment to the NR-responsive promoters are discussed.

Nucleosome sliding induced by the xMi-2 complex does not occur exclusively via a simple twist-diffusion mechanism.

ATP-dependent chromatin remodeling complexes can induce the translocation (sliding) of nucleosomes in cis along DNA, but the mechanism by which sliding occurs is not well defined. We previously presented evidence that sliding induced by the human SWI/SNF complex does not occur solely via a proposed "twist-diffusion" mechanism whereby the DNA rotates about its helical axis without displacement from the surface of the nucleosome (Aoyagi, S., and Hayes, J. J. (2002) Mol. Cell. Biol. 22, 7484-7490). Here we examined whether the Xenopus Mi-2 nucleosome remodeling complex induces nucleosome sliding via a twist-diffusion mechanism with nucleosomes assembled onto DNA templates containing branched DNA structures expected to sterically hinder rotation of the DNA helix on the nucleosome surface. We find that the branched DNA-containing nucleosomes undergo xMi-2-catalyzed sliding at a rate and extent identical to that of nucleosomes assembled on native DNA fragments. These results indicate that both the hSWI/SNF and xMi-2 complexes induce nucleosome sliding via a mechanism(s) other than simple twist diffusion and are consistent with models in which the DNA largely maintains its rotational orientation with respect to the histone surface.

Flap endonuclease 1 efficiently cleaves base excision repair and DNA replication intermediates assembled into nucleosomes.

Flap Endonuclease 1 (FEN1) plays important roles both in DNA replication and in base excision repair (BER). However, in both processes FEN1 substrates are likely to be assembled into chromatin. In order to examine how FEN1 is able to work within chromatin, we prepared model nucleosome substrates containing FEN1-cleavable DNA flaps. We find that human FEN1 binds and cleaves such substrates with efficiencies similar to that displayed with naked DNA. Moreover, we demonstrate that both FEN1 and human DNA ligase I can operate successively on DNA within the same nucleosome. These results suggest that some BER steps may not require nucleosome remodeling in vivo and that FEN 1 activity during Okazaki fragment processing can occur on nucleosomal substrates.