Cell Biology & Molecular Genetics

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    Impaired LEF1 Activation Accelerates iPSC-Derived Keratinocytes Differentiation in Hutchinson-Gilford Progeria Syndrome
    (MDPI, 2022-05-14) Mao, Xiaojing; Xiong, Zheng-Mei; Xue, Huijing; Brown, Markus A.; Gete, Yantenew G.; Yu, Reynold; Sun, Linlin; Cao, Kan
    Hutchinson–Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in the human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. Here, we studied the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, we investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected β-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. A chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to the early-stage epithelial developmental markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS.
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    DIVERGENT ROLES OF THE TCF4 AND LEF1 WNT SIGNALING TRANSCRIPTION FACTORS IN COLON CANCER
    (2021) Brown, Markus; Cao, Kan; Ried, Thomas; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The canonical WNT signaling pathway is necessary for guiding cell growth during embryonic development. In adults, WNT signaling maintains tissue stem cells and therefore plays an essential role in tissue homeostasis. In the colon, the WNT transcription factor, TCF4, is necessary for maintaining the intestinal stem cells. The initiating event in colon cancer is the aberrant activation of the WNT signaling pathway, which results in constitutive activity of TCF4. To determine how TCF4 influences colon cancer cell behavior, we silenced TCF7L2, the gene encoding TCF4, and used RNA sequencing and Hi-C to measure changes in transcription and nuclear structure in the SW480 colon cancer cell line. Loss of TCF4 resulted in A/B compartment switching, local chromatin reorganization, and a dramatic up-regulation in transcription. However, A/B compartment switching was not associated with changes in gene expression. We also found that loss of TCF4 resulted in the up-regulation of LEF1, another WNT transcription factor. Expressed LEF1 isoforms were found to be transcriptionally competent and over-compensated for WNT signaling activity upon loss of TCF4, suggesting a WNT-intrinsic feedback mechanism. Over-expression of LEF1 altered WNT signaling output to favor the expression of lymphoid genes, as opposed to a TCF4-based transcriptional program. ChIP-seq demonstrated that TCF4 and LEF1 bind distinct target genes, though they synergize to express MYC. TCF4 was found to bind the LEF1 promoter, indicative of direct repression, though LEF1 did not bind the TCF7L2 promoter. The CtBP1 protein, a known binding partner of TCF4, was found to be the most potent repressor of LEF1 expression. This demonstrates that despite the overall activation of WNT signaling in colon cancer, repressive functions of the WNT transcription factors are still intact, and the repression of LEF1 by TCF4 maintains a TCF4-centric transcriptional program in colon cancer cells.