The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes

The chromatin remodelling complex B-WICH, which comprises the William syndrome transcription factor (WSTF), SNF2h, and nuclear myosin 1 (NM1), is involved in regulating rDNA transcription, and SiRNA silencing of WSTF leads to a reduced level of 45S pre-rRNA. The mechanism behind the action of B-WICH is unclear. Here, we show that the B-WICH complex affects the chromatin structure and that silencing of the WSTF protein results in a compaction of the chromatin structure over a 200 basepair region at the rRNA promoter. WSTF knock down does not show an effect on the binding of the rRNA-specific enhancer and chromatin protein UBF, which contributes to the chromatin structure at active genes. Instead, WSTF knock down results in a reduced level of acetylated H3-Ac, in particular H3K9-Ac, at the promoter and along the gene. The association of the histone acetyl-transferases PCAF, p300 and GCN5 with the promoter is reduced in WSTF knock down cells, whereas the association of the histone acetyl-transferase MOF is retained. A low level of H3-Ac was also found in growing cells, but here histone acetyl-transferases were present at the rDNA promoter. We propose that the B-WICH complex remodels the chromatin structure at actively transcribed rRNA genes, and this allows for the association of specific histone acetyl-transferases

Gene regulation by nuclear hormone receptors in vivo

The nuclear hormone receptor superfamily contains 49 different members, including those for thyroid hormone, TRá and TRâ. These receptors are ligand-modulated transcription factors that activate or repress gene activity. The TRá gene encodes two main splice variants, TRá1 and TRá2. Furthermore, the orphan receptor Rev-erbAá gene is located juxtaposed to and partially overlapping the TRá gene, which has led to the hypothesis that transcription of one of them could interfere with expression of the other. In this thesis work I have studied the consequences for target gene expression and normal physiology of targeting the TR or the rev-erbAá genes in the mouse genome. The targeting of the Rev-erbAá gene by insertion of a â-gal cassette resulted in the appearance of novel transcripts for both Rev-erbAá and TR. Despite this, the TRá1 to TRá2 expression ratio was unaltered. On the other hand, the Rev-erbAá gene was found to negatively regulate its own expression, as shown by the â-gal activity in homozygote tissue being eight times higher than it is in heterozygotes and wt. Deletion of Rev-erbAá was also associated with a delayed cerebellar maturation. The results show that Rev-erbAá expression is important for an appropriate balance between transcriptional activation and repression during postnatal cerebellar development. TRá2 -deficient mice were generated using a different targeting approach: introduction of a strong polyadenylation site that blocks transcription into the TRá2 coding exon. This generated a shorter TRá1 transcript that was, however, expressed at increased levels. Both TRá2+/- and -/- mice had a complex phenotype with both hypothyroid and hyperthyroid features, attributable to the elevated expression of TRá1. This suggested that the ratio of TRá1 to TRá2 is important for maintaining an adequate physiological response to TH. TR ablation was shown to affect the regulation of several T3 responsive genes in the heart. Myosin heavy chain â expression, which normally is suppressed by T3-bound TR, was highly overexpressed in TRá1 -/- mice. However, it was suppressed by T3 in both TRá1 -/- and TRâ -/- mice, indicating that both isoforms can mediate repression. In contrast, the expression of MyHCá was not significantly affected by the deletion of either TRá or TRâ. Thus we conclude that the TR isoforms have distinct specificities in gene regulation of the cardiac MyHC genes. In our study of how T3 affects cardiac function, we found that the KCNE1 ion channel gene is negatively regulated by T3-activated TRá1. Transgenic mice overexpressing KCNE1 via a heart-specific promoter had very similar cardiac abnormalities to those of the TRá1 -/- mice. This supports the hypothesis that dysregulation of the KCNE1 gene can cause the cardiac dysfunction of TRá1 mice. Electrophysiological experiments confirmed that elevated KCNE1 levels perturb ion current activities, suggesting that the stoichiometry of the KCNE1 âsubunit relative to other ion channel á-subunits is of vital importance for heart function

Identification of Rev-erbalpha as a physiological repressor of apoC-III gene transcription.

Elevated serum levels of triglyceride-rich remnant lipoproteins (TRL) are a major risk factor predisposing a subject to atherosclerosis. Apolipoprotein C-III (apoC-III) is a major constituent of TRL that impedes triglyceride hydrolysis and remnant clearance and, as such, may exert pro-atherogenic activities. In the present study, transient cotransfection experiments in rat hepatocytes in primary culture and rabbit kidney RK13 cells demonstrated that overexpression of Rev-erbalpha specifically decreases basal and HNF-4 stimulated human apoC-III promoter activity. A Rev-erbalpha response element was mapped by promoter deletion, mutation analysis, and gel-shift experiments to a AGGTCA half-site located at position -23/-18 (downstream of the TATA box) in the apoC-III promoter. Finally, Rev-erbalpha-deficient mice displayed elevated serum and liver mRNA levels of apoC-III together with increased serum VLDL triglycerides. Taken together, our data identify Rev-erbalpha as a regulator of apoC-III gene expression, providing a novel, physiological role for this nuclear receptor in the regulation of lipid metabolism.info:eu-repo/semantics/publishe