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The C-terminus of the retinal homeobox (rax) gene product modulates transcription in a context-dependent manner.

ABSTRACT: Purpose:The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function. Methods:N-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene. Results:Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation. Conclusions:The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.

SUBMITTER: Buescher JL

PROVIDER: S-EPMC6386513 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The C-terminus of the <i>retinal homeobox (rax)</i> gene product modulates transcription in a context-dependent manner.

Buescher Jessica L JL El-Hodiri Heithem M HM

Molecular vision 20190223

<h4>Purpose</h4>The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The <i>rax</i> gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) doma ...[more]

PMID: 30820152

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Project description:Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional transcription factor that is essential for lytic HCMV infection. IE2 functions as an activator of viral early genes, negatively regulates its own promoter, and is required for viral replication. The mechanisms by which IE2 executes these distinct functions are incompletely understood. Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-chromatin immunoprecipitation (DFF-ChIP; employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP) approach that resolves occupancy and local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms that involve direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within their core promoter regions and blocking the assembly of preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 stimulates transcription by binding nearby, but not within, core promoter regions. In addition, IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 appears to be important for the synthesis of a spliced viral RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 does not functionally engage the host genome. Our results reveal mechanisms of transcriptional control by IE2, uncover a previously unknown function of IE2 as a Pol II elongation modulator, and demonstrate that DFF-ChIP is a useful tool for probing transcription factor occupancy and interactions between transcription factors and nucleosomes at high resolution. IMPORTANCE HCMV infects more than half of the world population and persists lifelong in its hosts. Although generally asymptomatic, HCMV infection can lead to life-threating disease in immunosuppressed individuals. Moreover, HCMV is the leading infectious cause of birth defects in the United States. As there are no vaccines effective against HCMV and antiviral drugs exhibit toxicity and are undermined by resistant HCMV variants, other vulnerabilities in HCMV must be explored. Here, we characterize the mechanism by which IE2 controls transcription during late HCMV infection. We demonstrate that IE2 engages numerous consensus sites across the HCMV genome and functions as an activator, repressor, or elongation modulator depending on the context of IE2 binding sites in relation to Pol II initiation and elongation complexes. Our findings have important implications for the ongoing exploration of IE2 as an antiviral drug target.

Dataset Information

The C-terminus of the retinal homeobox (rax) gene product modulates transcription in a context-dependent manner.

Publications

The C-terminus of the <i>retinal homeobox (rax)</i> gene product modulates transcription in a context-dependent manner.

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