Complex cardiac Nkx2-5 gene expression activated by noggin-sensitive enhancers followed by chamber-specific modules

Abstract

We previously reported that an Nkx2-5-GFP bacterial artificial chromosome in transgenic mice recapitulated the endogenous gene activity in the heart. Here, we identified three additional previously uncharacterized distal enhancer modules of Nkx2-5: UH6, which directed transgene expression in the right ventricle, interventricular septum, and atrial ventricular canal; UH5, which directed expression in both atria; and UH4, which directed transgene expression in tongue muscle. Nkx2-5 enhancers drive cardiogenic gene activity from the earliest progenitors to the late-stage embryonic heart, reside within its 27 kb of 5 flanking sequences, organized in a tandem array. Nkx2-5 enhancers involved with stomach-, tongue-, and chamber-restricted expression displayed lacZ transgene activity and chromatin histone acetylation patterns consistent with tissue-specific expression. An examination of Nkx2-5 gene activity in murine embryonic stem cells converted to beating embryoid bodies showed that only the proximal active region 2 and GATA-Smad enhancers were chromatin-remodeled. Chromatin remodeling of active region 2 and GATA-Smad enhancers were blunted by noggin coexpression, which indicated dependence on bone morphogenetic protein signaling for their chromatin activation during activation of Nkx2-5 expression.enhancer modules ͉ heart development ͉ Nkx2-5 gene regulation

T-box 2, a mediator of Bmp-Smad signaling, induced hyaluronan synthase 2 and Tgfβ2 expression and endocardial cushion formation

“…Deletion constructs were generated by restriction endonuclease digestion. The region from Ϫ2916 to Ϫ2602bp, containing multiple Smad sites, was linked to the hsp68lacZ reporter gene (29). Mutations at multiple Smad sites in Ϫ2916/ Ϫ2602 fragment were inserted using In-Fusion PCR cloning kit (Clontech) ( Fig.…”

“…Transfections were performed using Lipofectamine (Invitrogen) as described (30). Luciferase activity were measured using a luminometer to detect activates substrates, then normalized by ␤-galactosidase activity (29).…”

Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment

“…5I, white arrowheads) and harbors two well-defined enhancers: an early cardiac-specific enhancer at À9 kb from the start site of transcription and a stomach, pharynx, and thyroid-specific enhancer at À2.8 kb that are individually capable of directing tissue-appropriate expression in transgenic mice (Lien et al 1999;Reecy et al 1999). The cardiomyocyte enhancer recapitulates the expression pattern of the endogenous gene in cardiogenic precursors from the onset of lineage specification to looping heart tube, after which more distally located enhancers are needed to direct expression in the heart (Lien et al 1999;Reecy et al 1999;Chi et al 2005). The signals that make the À9-kb enhancer accessible during lineage commitment are unknown, although once open, its activity depends on Gata4 (Lien et al 1999;He et al 2011).…”

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

“…Thus polymorphisms have been mapped on physical maps of chromosomes using the technology [40,41]. Long-range regulatory elements of the mouse Nkx2-5 gene have also been mapped functionally using end-deleted EGFP functionalized BACs to generate transgenic mice [63]. More recently it has been used in enhancer-trapping using BACs.…”