Helicase-Like Transcription Factor (Hltf) Regulates G2/M Transition, Wt1/Gata4/Hif-1a Cardiac Transcription Networks, and Collagen Biogenesis

Abstract

HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum) brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf’s regulation of the G2/M transition (p=9.726E-15) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2) and lysyl (Plod2) collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3) for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus, silencing Hltf during heart organogenesis compromised DNA double-strand break repair, and caused aberrant collagen biogenesis altering the structural network that transmits cardiomyocyte force into muscle contraction.

The helicase-like transcription factor (HLTF) in cancer: loss of function or oncomorphic conversion of a tumor suppressor?

“…These findings underscore a role for HLTF in development (particularly for brain and heart), consistent with the findings of Gong et al, showing that HLTF transcripts were expressed in these organs [8]. The brain and heart transcriptomes were studied after the specific inactivation of Hltf in mice [23,27] and suggested additional roles for HLTF in different pathways ( Table 2). HLTF is involved in the G2/M transition of the cell cycle via 4 mechanisms: sister chromatid holding (HLTF deletion reduces the mRNAs of genes involved in chromosome cohesion), chromosome condensation (compaction of sister chromatids), centrosome separation and aberrant chromosome segregation and cell cycle arrest (resulting from HLTF deletion).…”

“…The protein forms (116 and 97 kDa, respectively) encoded by these alternatively spliced transcripts were both expressed in the brain, but only the fulllength protein (116 kDa) was expressed in the heart [27]. These findings underscore a role for HLTF in development (particularly for brain and heart), consistent with the findings of Gong et al, showing that HLTF transcripts were expressed in these organs [8].…”

“…HLTF was first identified as a transcription factor by identification of its target genes, its DNA-binding sequences and its protein partners [8,21,25,29,32] in different species and by analysis of heart and brain transcriptomes from a Hltf null mouse [23,27] (Tables 1, 2).…”

“…The only difference was that the screening was performed using either the HLTF DNA-binding domain expressed in E. coli as a glutathione-S-transferase (GST) fusion [32] or HLTF in rabbit nuclear extracts [33]. HLTF binding to these sequences was confirmed through sitedirected mutagenesis and luciferase assays [32] and through chromatin immunoprecipitation (ChIP; [21,23,25,27,[33][34][35][36]). …”

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

“…HLTF is an enzyme that stabilizes and repairs stalled replication forks and controls G2/M transition (49,59,60). Considering HLTF activities led us to investigate its role in Vpr-mediated G2 arrest.…”