Patterns of histone acetylation suggest dual pathways for gene activation by a bifunctional locus control region.

The five genes of the human growth hormone (hGH) cluster are expressed in either the pituitary or placenta. Activation of the cluster is dependent on a locus control region (LCR) comprising pituitary- specific (HSI,II, -15 kb), placenta-specific (HSIV, -30 kb) and shared (HSIII, -28 kb; HSV, -32 kb) DNase I hypersensitive sites. Gene activation in the pituitary is paralleled by acetylation of a 32 kb chromatin domain 5' to the cluster centered at HSI,II. In the present study we observed that acetylation of this region in placental chromatin was discretely limited to shared HSIII and HSV. Transgenic studies revealed placenta-specific activation of linked genes by a determinant (P-element) located 2 kb 5' to each of the four placentally expressed genes. A localized peak of histone acetylation was observed at these P-elements in placenta but not pituitary. These data support a model for bifunctional action of the hGH LCR in which separate positive determinants, HSI,II and the P-elements, activate their respective target genes by tissue-specific recruitment of distinctly regulated histone acetyl transferase activities.

Targeted recruitment of histone acetyltransferase activity to a locus control region.

Locus control regions (LCRs) are capable of activating target genes over substantial distances and establishing autonomously regulated chromatin domains. The basis for this action is poorly defined. Human growth hormone gene (hGH-N) expression is activated by an LCR marked by a series of DNase I-hypersensitive sites (HSI-III and HSV) in pituitary chromatin. These HSs are located between -15 and -32 kilobases (kb) relative to the hGH transcription start site. To establish a mechanistic basis for hGH LCR function, we carried out acetylation mapping of core histones H3 and H4 in chromatin encompassing the hGH cluster. These studies revealed that the entire LCR was selectively enriched for acetylation in chromatin isolated from a human pituitary somatotrope adenoma and in pituitaries of mice transgenic for the hGH locus, but not in hepatic or erythroid cells. Quantification of histone modification in the pituitary revealed a dramatic peak at HSI/II, the major pituitary-specific hGH LCR determinant (-15 kb), with gradually decreasing levels of modification extending from this site in both 5'- and 3'-directions. The 5'-border of the acetylated domain coincided with the 5' most hGH LCR element, HSV (-34 kb); and the 3'-border included the expressed hGH-N gene, but did not extend farther 3' into the placenta-specific region of the gene cluster. These data support a model of LCR function involving targeted recruitment and subsequent spreading of histone acetyltransferase activity to encompass and activate a remote target gene.

Tissue-specific expression of dominant negative mutant Drosophila HSC70 causes developmental defects and lethality.

The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins' dominant negative effects in vivo.

GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila.

We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS-lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported.