The THAP–zinc finger protein THAP1 regulates endothelial cell proliferation through modulation of pRB/E2F cell-cycle target genes

Abstract

We recently cloned a novel human nuclear factor (designated THAP1) from postcapillary venule endothelial cells (ECs) that contains a DNA-binding THAP domain, shared with zebrafish E2F6 and several Caenorhabditis elegans proteins interacting genetically with retinoblastoma gene product (pRB). Here, we show that THAP1 is a physiologic regulator of EC proliferation and cell-cycle progression, 2 essential processes for angiogenesis. Retroviral-mediated gene transfer of THAP1 into primary human ECs inhibited proliferation, and large-scale expression profiling with microarrays revealed that THAP1-mediated growth inhibition is due to coordinated repression of pRB/E2F cell-cycle target genes. Silencing of endogenous THAP1 through RNA interference similarly inhibited EC proliferation and G1/S cell-cycle progression, and resulted in down-regulation of several pRB/E2F cellcycle target genes, including RRM1, a gene required for S-phase DNA synthesis. Chromatin immunoprecipitation assays in proliferating ECs showed that endogenous THAP1 associates in vivo with a consensus THAP1-binding site found in the RRM1 promoter, indicating that RRM1 is a direct transcriptional target of THAP1. The similar phenotypes observed after THAP1 overexpression and silencing suggest that an optimal range of THAP1 expression is essential for EC proliferation. Together, these data provide the first links in mammals among THAP proteins, cell proliferation, and pRB/ E2F cell-cycle pathways. (Blood. 2007; 109:584-594)

Structure-Function Analysis of the THAP Zinc Finger of THAP1, a Large C2CH DNA-binding Module Linked to Rb/E2F Pathways

“…We found that human THAP1 is an endogenous physiological regulator of endothelial cell proliferation and G 1 /S cell cycle progression, which modulates expression of sev-eral pRb 4 /E2F cell cycle target genes. In addition, we identified RRM1, a G 1 /S-regulated gene required for S-phase DNA synthesis, as a direct transcriptional target of endogenous THAP1 (10). These data provided the first links in mammals between THAP proteins, cell proliferation, and pRB/E2F cell cycle pathways and complemented genetic data previously obtained in model animal organisms.…”

“…We recently demonstrated that THAP1 is a physiological regulator of cell proliferation. Silencing of THAP1 by RNA interference in human primary endothelial cells resulted in inhibition of G 1 /S cell cycle progression and down-modulation of several pRb/E2F cell cycle target genes, including RRM1, a gene activated at the G 1 /S transition and essential for S-phase DNA synthesis (10). We showed that the THAP zinc finger of THAP1 recognizes a consensus THAP1-binding site in the RRM1 promoter and that endogenous THAP1 associates in vivo with this site, indicating that RRM1 is a direct target gene of THAP1.…”

“…Electrophoresis was performed and gels were exposed as previously described (7). Supershift experiments were performed using 1 g of anti-THAP1 affinity-purified rabbit polyclonal antibodies (10).…”

“…Nevertheless, the C terminus displays structural variability. Indeed, the THAP domain of THAP1 shows two additional short 3 10 helices H3 and H4, encompassing residues 60 -63 and 79 -81, respectively, whereas the THAP domains of THAP2 and CtBP display additional two-stranded anti-parallel ␤-sheets. In the structure of the THAP domain of THAP2, the second anti-parallel ␤-sheet is formed by residues that would correspond to residues Phe 63 -Lys 64 and Leu 71 -Leu 72 in THAP1.…”

“…These results led us to search for the minimal size of the functional THAP zinc finger. The cysteine residues at the N terminus of the THAP domain (THAP1 Cys 5 and Cys 10 ) have previously been shown to be required for the functional activity of the domain, for both human THAP1 (7) and Drosophila P element transposase (41), thus defining the N-terminal boundary of the domain. In contrast, nothing was known about the requirements at the C terminus downstream of the conserved AVPTIF motif (6) containing the essential Pro 78 residue (7).…”