Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells.

Rett syndrome may be treated by reactivating the silent copy of Mecp2 from the inactive X chromosome in female cells. Most studies that model Mecp2 reactivation have used mouse fibroblasts rather than neural cells, which would be critical for phenotypic reversal, and rely on fluorescent reporters that lack adequate sensitivity. Here, we present a mouse model based on a dual bioluminescent and fluorescent reporter to assess the level of reactivation of Mecp2 and the inactive X chromosome by treating neural stem cells with 5-azacytidine and Xist knockdown. We show that reactivation of Mecp2 and other X-linked genes correlates with CpG density, with distance from escapees, and, very strongly, with the presence of short interspersed nuclear elements. In addition, X-linked genes reactivated in neural stem cells overlap substantially with early reactivating genes by induced pluripotent stem cell reprogramming of fibroblasts or neuronal progenitors, indicating that X chromosome reactivation follows similar paths regardless of the technique or cell type used.

Biology, Chemistry, and Pharmacology of Sirtuins.

Sirtuins are a family of protein deacylases related by amino acid sequence and cellular function to the yeast Saccharomyces cerevisiae protein Sir2 (Silent Information Regulator-2), the first of this class of enzymes to be identified and studied in detail. Based on its initially discovered activity, Sir2 was classified as a histone deacetylase that removes acetyl groups from histones H3 and H4. The acetylation/deacetylation of these particular substrates leads to changes in transcriptional silencing at specific loci in the yeast genome, hence its name. Sirtuins, however, have been shown to regulate a wide variety of cellular processes beyond transcriptional repression in varied subcellular compartments and in different cell types. Mechanistically distinct from Zn(2+)-dependent deacylases, sirtuins use nicotinamide adenine dinucleotide as a cofactor in the removal of acetyl and other acyl groups linking metabolic status and posttranslational modification. Sirtuins' unique position has made them attractive targets for small-molecule drug development. In this chapter, we describe the biological roles, therapeutic areas in which sirtuins may play a role and development of small-molecule inhibitors of sirtuins employing phenotypic screening technologies ranging from assays in yeast, as well as biochemical screens to yield lead drug development candidates targeting a broad spectrum of human diseases.

Identification of a small molecule inhibitor of Sir2p.

Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p, we identified a compound, splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA. In addition, whole genome microarray experiments show that splitomicin selectively inhibits Sir2p. In vitro, splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using splitomicin as a chemical genetic probe, we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells.

1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice.

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

Glucocorticoid-induced ketoacidosis in gestational diabetes: sequela of the acute treatment of preterm labor. A case report.

Pregnancy induces complex changes in energy metabolism, manifested clinically by insulin resistance, low fasting blood glucose levels, and proneness to ketosis. It is quite unusual for pregnant women who do not have type I diabetes to progress from ketosis to frank ketoacidosis, although this phenomenon is common in larger mammals. In the case described here, glucocorticoid administration in the setting of a prolonged fast triggered a metabolic cascade leading to ketoacidosis in a pregnant woman without type 1 diabetes. Other details of this illustrative case serve to synthesize several disparate observations regarding the pathogenesis of pregnancy ketoacidosis. Physicians should be aware of the potential for rapidly developing ketoacidosis with atypical biochemical and clinical features in pregnant women who are treated with high doses of glucocorticoids.

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice.

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

Differences in regulation of alpha 1(I) collagen promoter in vascular smooth muscle cells in transfection studies and transgenic mice.

It has previously been shown that the expression of a transgene containing 3.5 kb of alpha 1(l) collagen (COL1A1) promoter sequence fused to the chloramphenicol acetyl transferase (CAT) reporter gene (ColCAT3.6) paralleled the expression of the endogenous type I collagen gene in bone, tendon and skin whereas the expression in aorta was extremely low. In contrast, the same promoter construct showed comparable activity in a variety of transiently transfected smooth muscle and fibroblastic cells. In order to compare the activity of the transiently transfected and the ¿endogenous¿ transgene from a transgenic animal, in this study, vascular smooth muscle cells (VSMC) were isolated from ColCAT3.6 transgenic animals and used in a transfection assay. The endogenous transgene remained inactive in primary cultures of transgenic VSMC while transiently transfected ColCAT3.6 cells had comparable activity to NIH3T3 fibroblasts, primary rat tendon fibroblasts or primary rat VSMC. The immortalized cell line SM3T3, derived from the aorta of ColCAT3.6 transgenic mice did not express endogenous transgene but dis express stably transfected ColCAT3.6 to levels similar to NIH3T3 fibroblasts or osteoblastic ROS17/2.8 cells. This result stresses the influence of transgene history, possibly its passage through the germline, in regulation of tissue specific expression.

Identification of regulatory elements necessary for the expression of the COL1A1 promoter in murine odontoblasts.

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies). Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct. These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.

Regulation of type I collagen gene expression in bone.

The regulation of COL1A1 gene expression in bone was studied by measuring the activity of type I collagen promoter fusion genes (ColCAT) in permanently transfected osteoblastic cells and calvariae from transgenic animals. The basal activity of ColCAT fusion genes in transfected cells is mediated by DNA sequences between -3.5 to -2.3 kb while expression in vivo requires sequences between -2.3 and -1.7 kb. Parathyroid hormone, 1,25-dihydroxyvitamin D3 and interleukin-1 decrease the activity of ColCAT fusion genes in osteoblastic cells and transgenic calvariae. Because there may be differences between the expression of ColCAT fusion genes in cultured cells and intact bone, it will be important to compare data obtained from transfected cells with an in vivo model such as calvariae from transgenic mice.

Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells.

The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.

Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice.

The activity of fusion genes containing fragments of the COL1A1 promoter was measured in tissues from 6- to 8-day-old transgenic mice. ColCAT3.6 contains approximately 3.6 kb (-3521 to 115 bp) of the rat COL1A1 gene, the chloramphenicol acetyltransferase (CAT) reporter gene, and the SV40 splice and polyadenylation sequences. ColCAT2.3 and ColCAT1.7 are deletion constructs that contain 2296 and 1667 bp of COL1A1 upstream from the RNA start site, respectively. For each transgene, up to six lines of mice were characterized. Both ColCAT3.6 and ColCAT2.3 had similar activity in bone and tooth; ColCAT1.7 was inactive. In transgenic calvariae, levels of transgene mRNA paralleled levels of CAT activity. In tendon, the activity of ColCAT2.3 was 3- to 4-fold lower than that of ColCAT3.6, and the activity ColCAT1.7 was 16-fold lower than that of ColCAT2.3. There was little activity of the ColCAT constructs in liver and brain. These data show that DNA sequences between -2.3 and -1.7 kb are required for COL1A1 promoter expression in bone and tooth; sequences that control expression in tendon are distributed between -3.5 and -1.7 kb of the promoter, with sequences downstream of -1.7 kb still capable of directing expression to this tissue. The cis elements that govern basal expression of COL1A1 in transgenic calvariae appear to be different from those required for optimal expression of the COL1A1 promoter in stably transfected osteoblastic cells.

Regulation of the alpha 1(I) collagen promoter in vascular smooth muscle cells. Comparison with other alpha 1(I) collagen-producing cells in transgenic animals and cultured cells.

We have previously reported that the expression of the ColCAT3.6 transgene containing 3.5 kilobases (kb) of alpha 1(I) collagen (COL1A1) promoter sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene paralleled the expression of the endogenous gene in several connective tissues. We report here that the activity of the reporter gene in aorta from 7-day-old transgenic mice is 10-64-fold lower than in tendon or bone, whereas the endogenous gene is highly expressed in all three tissues. In contrast, the COL1A1 minigene containing 2.3 kb of upstream sequence, the first five exon/intron units, the last six exon/intron units, and 2 kb of 3'-flanking sequence showed high CAT activity in aorta. These results suggest that cis sequences found in ColCAT3.6 mediate high levels of COL1A1 expression in bone and tendon, but not in vascular smooth muscle cells (VSMC), whereas sequences located within the minigene, but not found in ColCAT3.6, mediate VSMC-specific expression. Analysis of promoter activity in cultured cells derived from transgenic tissues further suggests the presence of VSMC-specific regulatory domains. Transient transfection studies, however, failed to shows differential regulation. These differences stress the importance of not relying exclusively on transient transfection data when mapping tissue-specific regulatory domains.

Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.