Linda L. Pritchard

CBP/p300 and muscle differentiation: no HAT, no muscle

Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle‐specific and ubiquitous genes. Two distinct families of transcriptional co‐activators, GCN5/PCAF and CREB‐binding protein (CBP)/p300, are crucial to this process. While both possess histone acetyl‐transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. We have addressed this issue directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. Our results clearly demonstrate that CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program.

Cell cycle‐dependent recruitment of HDAC‐1 correlates with deacetylation of histone H4 on an Rb–E2F target promoter

The transcription factor E2F, which is a key element in the control of cell proliferation, is repressed by Rb and other pocket proteins in growth‐arrested differentiating cells, as well as in proliferating cells when they progress through early G1. It is not known whether similar mechanisms are operative in the two situations. A body of data suggests that E2F repression by pocket proteins involves class I histone deacetylases (HDACs). It has been hypothesized that these enzymes are recruited to E2F target promoters where they deacetylate histones. Here we have tested this hypothesis directly by using formaldehyde cross‐linked chromatin immunoprecipitation (XChIP) assays to evaluate HDAC association in living cells. Our data show that a histone deacetylase, HDAC‐1, is stably bound to an E2F target promoter during early G1 in proliferating cells and released at the G1–S transition. In addition, our results reveal an inverse correlation between HDAC‐1 recruitment and histone H4 acetylation on specific lysines.

Acetylation is important for MyoD function in adult mice

Acetylation is a post‐translational modification that influences the activity of numerous proteins in vitro. Among them, the myogenic transcription factor MyoD shows an increased transcriptional activity in vitro when acetylated on two lysines (K): lysines 99 and 102. Here, we have investigated the biological relevance of this acetylation in vivo. Using specific antibodies, we show that endogenous MyoD is acetylated on lysines 99 and 102 in myoblasts. Moreover, we show the functional importance of acetylation in live animals by using a mutant of MyoD in which lysines 99 and 102 were replaced by arginines (R). Knock‐in embryos homozygous for the MyoDR99,102 allele expressed slightly reduced levels of MyoD but developed normally. However, the knock‐in homozygous adult mice showed a phenotype that was almost identical to that of MyoD‐knockout animals, including delayed muscle regeneration in vivo and an increased number of myoblasts but with reduced differentiation potential in vitro. Together, these results show the importance of MyoD acetylation for adult myogenesis.

The WEE1 regulators CPEB1 and miR-15b switch from inhibitor to activators at G2/M

ABSTRACT MicroRNAs (miRNAs) in the AGO-containing RISC complex control messenger RNA (mRNA) translation by binding to mRNA 3′ untranslated region (3′UTR). The relationship between miRNAs and other regulatory factors that also bind to mRNA 3′UTR, such as CPEB1 (cytoplasmic polyadenylation element-binding protein), remains elusive. We found that both CPEB1 and miR-15b control the expression of WEE1, a key mammalian cell cycle regulator. Together, they repress WEE1 protein expression during G1 and S-phase. Interestingly, the 2 factors lose their inhibitory activity at the G2/M transition, at the time of the cell cycle when WEE1 expression is maximal, and, moreover, rather activate WEE1 translation in a synergistic manner. Our data show that translational regulation by RISC and CPEB1 is essential in cell cycle control and, most importantly, is coordinated, and can be switched from inhibition to activation during the cell cycle.

Small Regulatory RNAs and Skeletal Muscle Cell Differentiation

Until recently, RNA was considered to be merely a downstream effector of the “noble” genome, the latter having all the information and therefore occupying a position at the very heart of gene regulation, according to the “central dogma” of DNA transcribed into RNA translated into protein. Although we all knew that RNAs also have accessory functions, and that non-coding RNAs intervene at all stages of gene expression, these essential functions were considered to be mere “housekeepers,” and RNA was denied a regulatory role. This dogma was, however, “blown up” several years ago by the concomitant discovery of RNA interference and microRNAs in a model organism, the worm Caenorhabditis elegans. We now know that small regulatory RNAs are widely conserved in plants and animals, and that microRNAs and short interfering RNAs are not the only kinds of regulatory small RNAs that exist. Indeed, the variety of functions in which small non-coding RNAs have been shown to play essential roles has grown rapidly. Basically, they are involved in controlling large genetic programs or large regions of cell genomes, and they participate in determining what is called cell fate, or the balance between cell proliferation, differentiation and death. This equilibrium is strictly controlled under normal conditions, and its deregulation leads to oncogenesis. One of our main interests is the function of microRNAs in mammalian skeletal muscle. We describe here the high-throughput screening strategy used in our laboratory to identify and validate the microRNAs and their specific targets which are essential for muscle cell differentiation.