Touma, M. | bioRxiv Science

bioRxiv2025

Wnt11 Positively Regulates Neonatal Cardiomyocyte Maturation at the Interphase of Life via Frizzled 4 Receptor

Congenital heart defects (CHDs) affect 1% of live births and remain the leading cause of infant morbidity and early mortality. While most studies focus on the genetic basis of CHDs, relatively little is known about the interplay between intrinsic signaling and external environmental factors in the progression of CHDs after birth during the perinatal circulatory transition window when environmental stress factors are prevalent. We recently explored such interplay through a newly identified gene-environment regulatory circuit involving Wnt11 signaling and systemic hypoxia. Specifically, we demonstrated that activation of the Wnt11/Rb1 axis is critical for normal chamber-specific development after birth. This regulatory switch is disrupted by systemic hypoxia more robustly in the right ventricle (RV) than the left ventricle (LV), leading to enhanced neonatal cardiomyocyte cell cycle activity in an RV-specific manner, resulting in delayed maturation and attenuation of ventricular patterning in response to systemic hypoxia stress in the neonatal heart. Furthermore, we found that the Wnt11/Rb1 axis is also inactivated in infantile hearts with cyanotic CHDs, such as tetralogy of Fallot (TOF), potentially contributing to hypoxia-associated RV abnormalities in this context. However, the molecular players of this signaling cascade in neonatal cardiomyocyte remain largely unknown. Herein, we report that Frizzled 4 (Fzd4) acts as a specific upstream receptor for Wnt11 in neonatal cardiomyocytes. Specifically, Fzd4 exhibited an expression pattern like Wnt11 in neonatal heart perinatal circulatory transition under normal and hypoxemic environments. Furthermore, Fzd4 loss in neonatal cardiomyocytes stimulated cardiomyocyte cell cycle activity and disrupted the Wnt11-Rb1 signaling axis mirroring the impact of the Wnt11-deficient cardiomyocyte phenotype. Finally, co-immunoprecipitation analysis confirmed the Wnt11-Fzd4 binding in isolated neonatal cardiomyocytes and intact hearts. These results demonstrate that Fzd4 is a specific and required upstream receptor for the Wnt11-Rb1 signaling activity in the neonatal heart and provides mechanistic insights into the essential role of Wnt11 as a key positive regulator of neonatal cardiomyocyte transition from proliferative to mature phenotype at the interphase of life.

cell biology↗

bioRxiv2023

Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed an unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (P-value < 1e-5 and enrichment score [≥] 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA polII, including TATA, transcription initiator, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns, were enriched with the Homeobox family of transcription factors, and exhibited TA-rich motif sequences, suggesting potential motif specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers,12% exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may potentially dictate its function in myogenic differentiation and potentially other cellular and biological processes.

genomics↗