Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I-HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.

Two forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway.

The development of a complex organ requires the specification of appropriate numbers of each of its constituent cell types, as well as their proper differentiation and correct positioning relative to each other. During Drosophila cardiogenesis, all three of these processes are controlled by jumeau (jumu) and Checkpoint suppressor homologue (CHES-1-like), two genes encoding forkhead transcription factors that we discovered utilizing an integrated genetic, genomic, and computational strategy for identifying genes expressed in the developing Drosophila heart. Both jumu and CHES-1-like are required during asymmetric cell division for the derivation of two distinct cardiac cell types from their mutual precursor and in symmetric cell divisions that produce yet a third type of heart cell. jumu and CHES-1-like control the division of cardiac progenitors by regulating the activity of Polo, a kinase involved in multiple steps of mitosis. This pathway demonstrates how transcription factors integrate diverse developmental processes during organogenesis.

A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis.

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA-based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type-specific developmental gene expression patterns.

Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors.

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs - including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) - to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, our results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression.

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited.

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.

Hepatic ABCG5 and ABCG8 overexpression increases hepatobiliary sterol transport but does not alter aortic atherosclerosis in transgenic mice.

The individual roles of hepatic versus intestinal ABCG5 and ABCG8 in sterol transport have not yet been investigated. To determine the specific contribution of liver ABCG5/G8 to sterol transport and atherosclerosis, we generated transgenic mice that overexpress human ABCG5 and ABCG8 in the liver but not intestine (liver G5/G8-Tg) in three different genetic backgrounds: C57Bl/6, apoE-KO, and low density lipoprotein receptor (LDLr)-KO. Hepatic overexpression of ABCG5/G8 enhanced hepatobiliary secretion of cholesterol and plant sterols by 1.5-2-fold, increased the amount of intestinal cholesterol available for absorption and fecal excretion by up to 27%, and decreased the accumulation of plant sterols in plasma by approximately 25%. However, it did not alter fractional intestinal cholesterol absorption, fecal neutral sterol excretion, hepatic cholesterol concentrations, or hepatic cholesterol synthesis. Consequently, overexpression of ABCG5/G8 in only the liver had no effect on the plasma lipid profile, including cholesterol, HDL-C, and non-HDL-C, or on the development of proximal aortic atherosclerosis in C57Bl/6, apoE-KO, or LDLr-KO mice. Thus, liver ABCG5/G8 facilitate the secretion of liver sterols into bile and serve as an alternative mechanism, independent of intestinal ABCG5/G8, to protect against the accumulation of dietary plant sterols in plasma. However, in the absence of changes in fractional intestinal cholesterol absorption, increased secretion of sterols into bile induced by hepatic overexpression of ABCG5/G8 was not sufficient to alter hepatic cholesterol balance, enhance cholesterol removal from the body or to alter atherogenic risk in liver G5/G8-Tg mice. These findings demonstrate that overexpression of ABCG5/G8 in the liver profoundly alters hepatic but not intestinal sterol transport, identifying distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism.

Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations.

The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.