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

<div><p>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 <i>postpartum</i>) 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.</p> </div

Photomicrographs of control (A-F) and Hltf null (G-L) hearts.

<p>Sections were stained with picrosirius red and imaged with transmitted DIC microscopy and epifluorescence. DIC images (left column) were merged with strong green (intrinsic) fluorescence of cells and elastic fibers (middle column) to show picrosirius red-stained collagen (right column). Note the wavy collagen bundles in the controls (C, F), compared with tangled bundles (I) and thin, short disorganized fibers (L) in Hltf null hearts. Arrows and asterisks (*) are used to show collagen lacks intrinsic fluorescence.</p

The regulatory region of the Gata4 gene.

<p>Transcriptional regulation of Gata4 occurs at a 0.25 kb region upstream of the transcription start site (SS) [35]. Authentic regulatory sites, and putative Hltf binding sites (red) are underlined. Hltf binding to this region of the promoter was authenticated by ChIP-PCR (Figure 2, Panel B). Downregulation of Hif-1a protein binding (red) is predicted from reduced transcription of the Hif-1a gene in Hltf null heart.</p

Hltf regulates Gata4-dependent and Gata4-independent pathways.

<p>Hltf silencing resulted in the downregulation (threshold 1.1, p < 0.05) of previously unknown target genes (blue circles) and their direct targets (open circles).</p

Spectral imaging microscopy of intrinsic fluorescence of control (A-D) and Hltf null (E-L) hearts.

<p>Heart sections stained with picrosirius red were imaged using 488 excitation (mercury lamp) and a halogen lamp to obtain zero order spectra of regions exhibiting both elastin and collagen. Cells were imaged using a 60x oil objective (N.A. 1.4) and a 500 ms exposure. Images A, E, and I were obtained using a 3 μm slit width on the spectrograph. For images B, F and J, the slit width was closed to 1 μm. For images C, G and K the slit width was closed to 0.25 μm. This ensured the highest spatial resolution from a discrete area. Notice in A-C the highly ordered structure of wavy collagen is apparent (arrows 3 and 4). The areas corresponding to elastin were obtained from regions of interest (ROI) at arrows 1 and 2. The corresponding first order spectra at 0.4 nm resolution (D) shows the ROI labeled as 1 and 2 correspond to elastin with peaks at 515 and 520 nm; whereas collagen (arrows 3 and 4) shows an emission shoulder at 533 nm. Data were normalized since the intensity for spectra 1 and 2 were lower for elastin (14,000 arbitrary units of fluorescence, a.u.f.) than spectra 3 and 4 for collagen (a.u.f. 60,000). Images E-G and I-K with their corresponding spectra in H and L, respectively, are from null hearts. In E-G, the collagen is very thin and less structured than in controls (A-C). As shown in H, the emission spectra for collagen (a.u.f. 40,000) and elastin are similar to controls. Significant heterogeneity in collagen and elastin structure was also observed in null hearts (I-K). As shown in L, some cases of significant displacement in collagen structure were observed at 540 nm, along with a broader shoulder on elastin at 520-530 nm.</p

Western blot, ChIP-PCR and competitive RT-PCR.

<p>Panel A, Western blotting confirmed the presence of full-length Hltf protein (116-kDa between molecular markers 120 and 100 kDa) in hearts of control mice (lane 1), compared with the absence of protein expression in hearts of Hltf null mice (lane 2). Panel B, electrophoretic resolution of a single population of amplicons from touchdown PCR of ChIP confirmed Hltf bound the transcriptionally active regulatory regions of the promoters of Gata4 (248-bp; lane 1) and Myh7b/miR499 (228-bp; lane 2). Water blank control (lane 3) and molecular ladder (bp) are shown. Panel C, electrophoretic resolution of a single population of amplicons from touchdown PCR of ChIP confirmed Hltf bound the putative promoter of Hif-1a (600-bp, lane 1). Water blank control (lane 2) and φX174 markers are included. Panel D, analysis of competitive RT-PCR products by electrophoresis for the presence/absence of exon 7 in heart (lanes 1-2) and brain (lanes 3-4) revealed the ratio of amplicons without exon 7 (293-bp) to amplicons with exon 7 (390-bp) was the same in the presence (lane 1, 3) and absence (lane 2, 4) of Hltf. The water blank control (lane 5) and φX174 markers are added for completeness. The identity of each population of amplicons was verified by double-stranded sequencing.</p

Scatter plot of expression level measured by RPKM: control vs.

<div><p><b>Hltf knockout</b>. </p> <p>Panel A, each point is the mean of three replicate RPKM values for an individual gene in control heart plotted against values of the corresponding gene in Hltf null heart. The black line indicates the linear regression. R² = 0.9818, correlation coefficient. Panel B, genes with statistically significant expression (each dot is the mean of three replicate RPKM values, differential expression >1.1 fold, and P < 0.05) in control vs. null heart samples. Genes with specific importance to this study [Gata4, Myh7b, Wt1, Hif-1a] are labeled with a red dot and a black arrow.</p></div

Hltf null phenotype compared with controls at 6-8 hours postpartum.

<p>Compared with controls (A), Hltf null pups developed cyanosis (B). Deviation in heart shape between control (C) and Hltf null mice (D) are evident in H & E stained sections. Increased (p<0.0001) active caspase 3 (E) showed elevated apoptosis in Hltf null hearts. One of five null pups had a rare left coronary artery fistula (F).</p

The putative promoter of Hif-1a.

<p>Transcription and translation start sites (SS) from mm9 reference genome and NCBI reference sequence NM_010431.2. Putative Hltf binding sites (red) are underlined. Hltf binding to this regulatory region was authenticated by ChIP-PCR (Figure 2, Panel C).</p

Hltf null hearts (1) and brains (2) share the same defects in the G2/M transition.

<p>Significant decreases (red thermometers = negative effects) in transcript expression of major components of the G2/M transition in Hltf null heart (1) and brain (2) are superimposed on the proprietary multicomponent canonical pathway map from the MetaCore™ database [Straight lines = interactions; Symbols = events; +P = phosphorylation; B = binding; GR = group relation; CS = complex subunit. Colors for lines and symbols are green for positive, and gray for unspecified]. </p