Abstract
The molecular mechanisms underlying mesodermal and cardiac specification from embryonic stem cells (ESCs) are not fully understood. Here, we showed that the BTB domain-containing zinc finger protein CIBZ is expressed in mouse ESCs but is dramatically downregulated during ESC differentiation. CIBZ deletion in ESCs induced specification toward mesoderm phenotypes and their differentiation into cardiomyocytes, whereas overexpression of CIBZ delayed these processes. During ESC differentiation, CIBZ loss-and-gain-of-function data indicate that CIBZ negatively regulates the expressions of Brachyury (T) and Mesp1, the key transcriptional factors responsible for the specification of mammalian mesoderm and cardiac progenitors, respectively. Chromatin immunoprecipitation assays showed that CIBZ binds to T and Mesp1 promoters in undifferentiated ESCs, and luciferase assays indicate that CIBZ suppresses T and Mesp1 promoters. These findings demonstrate that CIBZ is a novel regulator of mesodermal and cardiac differentiation of ESCs, and suggest that CIBZ-mediated cardiac differentiation depends on the regulation of these two genes.
Highlights
Heart disease is one of the leading causes of death in the world[1,2]
SiRNA-mediated transient knockdown of CIBZ in embryonic stem cells (ESCs) led to the induction of T and Mesp[1] genes during ESC differentiation on days 3–5 (Supplementary Fig. S2)
Twofold forced expression of Oct3/4 in ESCs resulted in induction of T and Mesp[1], which have been associated with ESC commitment to mesodermal and cardiac lineage
Summary
Heart disease is one of the leading causes of death in the world[1,2]. Many heart diseases are caused by the massive loss or dysfunction of cardiomyocytes[2]. Mesp[1] alone has been shown to sufficiently induce cardiac specification by positively regulating downstream target genes, such as the markers of cardiac progenitors (Nkx2.5), first heart fields (Tbx5), and second heart fields (Isl1)[10,11]. Subsequent cardiac differentiation leads to the development of cardiac progenitor cells into mature cardiomyocytes that express structural proteins, including the cardiac troponins I and T (cTnI and cTnT) and myosin heavy chain (MHC)[12]. We identified a BTB domain-containing zinc finger protein (CIBZ; ZBTB38 in humans) that is ubiquitously expressed in mouse tissues and in various cell lines[14,15]. CIBZ induces apoptosis in murine cells[16], and that CIBZ negatively regulates myogenic differentiation by binding and inhibiting the myogenin promoter via its zinc finger domains[17]. Because CIBZ is highly expressed in ESCs and is critical for ESC proliferation[18], we hypothesized that CIBZ plays a role in the regulation of ESC differentiation
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