The histone methyltransferase MLL is an upstream regulator of endothelial-cell sprout formation

Abstract

Posttranslational histone modification by acetylation or methylation regulates gene expression. Here, we investigated the role of the histone lysine methyltransferase MLL for angiogenic functions in human umbilical vein endothelial cells. Suppression of MLL expression by siRNA or incubation with the pharmacologic methyltransferase inhibitor 5-deoxy-5-(methylthio)adenosine significantly decreased endothelial-cell migration and capillary sprout formation, indicating that methyltransferase activity is required for proangiogenic endothelialcell functions. Because the expression of homeodomain transcription factors (Hox) is regulated by MLL, we elucidated the role of Hox gene expression. MLL silencing was associated with reduced mRNA and protein expression of HoxA9 and HoxD3, whereas HoxB3, HoxB4, HoxB5, and HoxB9 were not altered. Overexpression of HoxA9 or HoxD3 partially compensated for impaired migration in MLL siRNA-transfected endothelial cells, suggesting that HoxA9 and HoxD3 both contribute to MLL-dependent migration. As a potential underlying mechanism, MLL siRNA down-regulated mRNA and protein levels of the HoxA9-dependent axon guidance factor EphB4. In contrast, MLL knockdown effects on capillary sprouting were not rescued by HoxA9 or HoxD3 overexpression, indicating that MLL affects additional targets required for 3-dimensional sprout formation. IntroductionMLL (mixed lineage leukemia, ALL-1, HRX) is a chromatin regulator homolog to the Drosophila trithorax gene. 1-3 MLL was originally discovered by its involvement in human acute leukemia through chromosomal translocation and fusion to a variety of genes. 4 MLL fusion proteins were shown to cause myeloid progenitor immortalization by inducing a characteristic expression profile of homeobox (Hox) transcription factors. 5,6 Physiologically, the SET domain of MLL catalyzes the methylation of lysine 4 of histone H3, thereby regulating gene expression. 7,8 In particular, native MLL is essential for the epigenetic control of gene expression of a set of homeobox proteins HoxA7, HoxA9, and HoxA10 during ex vivo differentiation of embryonic stem cells 9 and for physiologic hematopoiesis. 10-12 MLL Ϫ/Ϫ mice die around dpc 10.5 with aberrant rostrocaudal segmentation, 13 or, in a different model targeting exons 12 to 14, at dpc 11.5 to 14.5 with edematous bodies and petechiae. 12 Beyond segmental identity and hematopoiesis, MLL is crucial for the maintenance of helper T-lymphocyte function. 14 As yet, however, a function of MLL in postnatal angiogenesis has not been explored.Hox transcription factors play important roles during embryonic development of the cardiovascular system. [15][16][17] Certain members of the Hox family also regulate postnatal angiogenesis and endothelialcell adhesion and migration. HoxD3 promotes endothelial invasion and migration 18 and converts resting endothelium to an angiogenic phenotype by inducing proangiogenic genes including the integrin subunits ␣5 and ␤3. 19 HoxB3 is required for capillary morphogenesis of preformed vascular sprout...

BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression

“…shRNA knockdown of SET1A had no effect on p21 or p14 expression (see Fig. S12A in the supplemental material), suggesting that depletion of SET1A decreases the expression of specific genes while having no effect on others, as has been shown by others (8). ChIP with an anti-H3K4 antibody followed by QPCR with primers overlapping the upstream promoter regions p21 and p14, but not adjacent to any nearby CTCF binding site, also showed no change in histone dimethylation (see Fig.…”

Histone Deacetylase 9 Promotes Angiogenesis by Targeting the Antiangiogenic MicroRNA-17–92 Cluster in Endothelial Cells

“…32 In vitro angiogenesis was quantified by measuring the cumulative length of the sprouts, the number of sprouts, and the number of branch points that had grown out of each spheroid using a digital imaging software (Axiovision 4.6, Carl Zeiss Imaging Solutions GmbH, Munich, Germany) analyzing 10 spheroids per by guest on May 9, 2018 http://atvb.ahajournals.org/ Downloaded from group and experiment. To measure tube formation, 75 000 HUVECs were seeded in 1 mL of endothelial basal medium (10% FCS) on the Matrigel Basement Membrane Matrix, as described previously.…”

The synergistic regulation of VEGF-mediated angiogenesis through miR-190 and target genes

“…Endothelial cell spheroids of a defined cell number were generated as described previously (Korff and Augustin 1998;Diehl et al 2007). In vitro angiogenesis was quantified by measuring the cumulative length of the sprouts that had grown out of each spheroid using ImageJ software analyzing 10 spheroids per group and experiment.…”