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.
Highlights
Congenital heart defects (CHDs) - 1 in 100 newborns - result from errors in the development of heart valves, chambers and blood vessels [1]
The resulting helicase-like transcription factor (Hltf) null mice have Hltf deleted from all tissues
Hltf controls the expression of key components of the G2/M transition as well as the Fanconi Anemia (FA)/Brca pathway in DNA repair, and cell cycle checkpoint
Summary
Congenital heart defects (CHDs) - 1 in 100 newborns - result from errors in the development of heart valves, chambers and blood vessels [1]. Transgenic and knockout mouse models have helped to characterize transcription factors (MEF2, GATA4, SRF, NFkB, and NFAT) that regulate the fetal cardiac gene program [3]. These transcription factors participate in the reactivation of the fetal cardiac gene program in response to physiological stress. EPDCs differentiate into a variety of cell types including cardiac fibroblasts that are responsible for synthesis of ECM, a scaffold for fibroblast-myocyte-capillary interactions [6]. Our recent work with a helicase-like transcription factor (Hltf) null mouse model indicates Hltf has an important role in cardiac morphogenesis through its regulation of Gata and Wt1. Hltf regulates homeostasis of the three-dimensional ECM collagen scaffold via its transcriptional control of Hif-1a
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