Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loophelix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase B␣ relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner.Cardiac transcription factors play essential roles in regulating tissue-specific gene expression during proper development and function of the heart (1-3). The expression profiles of distinct sets of cardiac genes are altered in cardiac disease, indicating the importance of transcriptional regulation in response to disease stimuli (1-3). Transcription factors form multiprotein complexes, and combinatorial actions of transcription factors in such complexes dictate the specificity of downstream gene expression. For example, the physical and functional interaction among various cardiac transcription factors including MEF2, NKX2.5, TBX5, and GATA4 can be regulated by upstream cellular signaling and is likely to be impaired in patients with congenital heart disease (4 -7).Members of the Hairy-related transcription factor (HRT) 1 family of repressors, also known as Hesr, Hey, CHF, gridlock, and HERP (8 -13), are highly expressed in the heart and vasculature. HRT proteins have a basic helix-loop-helix (bHLH) motif, an orange domain, and a conserved C-terminal tetrapeptide motif and show the highest structural similarity to Hairy and Enhancer of split in flies and the mammalian HES family proteins (8 -13). Expression of the HRT genes is activated by Notch signaling, suggesting a role for HRT proteins as transcriptional mediators of Notch signaling in the cardiovascular system (14 -17). Mutations of the HRT2 ortholog in zebrafish, gridlock, result in defects of aortic development, and misexpression of gridlock favors the development of arteries over veins (12,17). In mice, targeted disruption of Hrt2/Hey...

Silencing of the cryptic mating-type locus HMR requires recognition of a small DNA sequence element, the HMR-E silencer, by the Sir1p, one of four Sir proteins required for the assembly of silenced chromatin domains in Saccharomyces cerevisiae. The Sir1p recognizes the silencer through interactions with the origin recognition complex (ORC), a protein complex that binds the silencer DNA directly. Sir1p was physically associated with HMR in chromatin, and this association required a Sir1p-ORC interaction, suggesting that it reflected the Sir1p silencer-recognition function required for silencing. Sir1p was not associated with nonsilencer replication origins that bind the ORC, indicating that a Sir1p-ORC interaction is confined to silencers. Significantly, the other SIR genes were required for Sir1p's association with HMR. Thus, multiple protein contacts required for and unique to silent chromatin may confine a Sir1p-ORC interaction to silencers. The Sir1p was present at extremely low concentrations in yeast cells yet was associated with HMR at all stages of the cell cycle examined. These data provide insights into the mechanisms that establish and restrict the assembly of silenced chromatin to only a few discrete chromosomal domains.

In fission yeast and multicellular organisms, centromere-proximal regions of chromosomes are heterochromatic, containing proteins that silence gene expression. In contrast, the relationship between heterochromatin proteins and kinetochore function in the budding yeast Saccharomyces cerevisiae remains largely unexplored. Here we report that the yeast heterochromatin protein Sir1 is a component of centromeric chromatin and contributes to mitotic chromosome stability. Sir1 recruitment to centromeres occurred through a novel mechanism independent of its interaction with the origin recognition complex (ORC). Sir1 function at centromeres was distinct from its role in forming heterochromatin, because the Sir2-4 proteins were not associated with centromeric regions. Sir1 bound to Cac1, a subunit of chromatin assembly factor I (CAF-I), and helped to retain Cac1 at centromeric loci. These studies reveal that although budding yeast and mammalian cells use fundamentally different mechanisms of forming heterochromatin, they both use silencing proteins to attract the histone deposition factor CAF-I to centromeric chromatin.Supplemental material is available at http://www.genesdev.org.

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Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes.