Bruna P. Brylawski

Microbial Regulation of Glucose Metabolism and Cell-Cycle Progression in Mammalian Colonocytes

A prodigious number of microbes inhabit the human body, especially in the lumen of the gastrointestinal (GI) tract, yet our knowledge of how they regulate metabolic pathways within our cells is rather limited. To investigate the role of microbiota in host energy metabolism, we analyzed ATP levels and AMPK phosphorylation in tissues isolated from germfree and conventionally-raised C57BL/6 mice. These experiments demonstrated that microbiota are required for energy homeostasis in the proximal colon to a greater extent than other segments of the GI tract that also harbor high densities of bacteria. This tissue-specific effect is consistent with colonocytes utilizing bacterially-produced butyrate as their primary energy source, whereas most other cell types utilize glucose. However, it was surprising that glucose did not compensate for butyrate deficiency. We measured a 3.5-fold increase in glucose uptake in germfree colonocytes. However, 13C-glucose metabolic-flux experiments and biochemical assays demonstrated that they shifted their glucose metabolism away from mitochondrial oxidation/CO2 production and toward increased glycolysis/lactate production, which does not yield enough ATPs to compensate. The mechanism responsible for this metabolic shift is diminished pyruvate dehydrogenase (PDH) levels and activity. Consistent with perturbed PDH function, the addition of butyrate, but not glucose, to germfree colonocytes ex vivo stimulated oxidative metabolism. As a result of this energetic defect, germfree colonocytes exhibited a partial block in the G1-to-S-phase transition that was rescued by a butyrate-fortified diet. These data reveal a mechanism by which microbiota regulate glucose utilization to influence energy homeostasis and cell-cycle progression of mammalian host cells.

Same origins of DNA replication function on the active and inactive human X chromosomes

We previously characterized a functional origin of DNA replication at the transcriptional promoter of the human hypoxanthine‐guanine phosphoribosyltransferase (HPRT) gene (Cohen et al. [ 2002 ] J. Cell. Biochem. 85:346‐356). This origin was mapped using a quantitative PCR assay to evaluate the relative abundance of HPRT markers in short nascent DNA strands isolated from asynchronous cultures of male fibroblasts. The HPRT gene on the X chromosome is transcriptionally active in male human fibroblasts. It is known that on the heterochromatic X chromosome in female cells the HPRT gene is transcriptionally silenced and its replication timing changes from early to late in S phase. This change in replication timing could indicate that replication of the HPRT gene is under the control of different origins of DNA replication in the active (euchromatic, early replicating) and the inactive (heterochromatic, late replicating) X chromosomes. In the present study, we identified the location of the origin of replication of a second X chromosome gene, glucose‐6‐phosphate dehydrogenase (G6PD), which we mapped to its transcriptional promoter, in normal male human fibroblasts. Then, we determined the activity of the previously identified HPRT and the G6PD human origins in hybrid hamster cells carrying either the active or the inactive human X chromosome. The results of these studies clearly demonstrated that the human HPRT and G6PD origins of replication were utilized to the same extent in the active and the inactive X chromosomes. Therefore, transcription activity at the HPRT and G6PD genes is not necessary for initiation of DNA replication at the origins mapped to these chromosomal loci. J. Cell. Biochem. 88: 923–931, 2003.

Mapping of an origin of DNA replication near the transcriptional promoter of the human HPRT gene

A quantitative PCR method was used to map a functional origin of DNA replication in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. This PCR method measures the abundance of specific sequences in short fragments of newly replicated DNA from logarithmically growing cells. Quantitative measurements rely on synthetic molecules (competitors) that amplify with the same primer sets as the target molecules, but generate products of different sizes. This method was first utilized to determine the position of the replication origin near the lamin B2 gene (Giacca et al. [ 1994 ] Proc. Natl. Acad. Sci. U S A. 91:7119–7123). In the present study, primer sets were tested along a 16‐kb region near exon 1 of the HPRT gene. The most abundant fragment was found to be located in the first intron of HPRT, just downstream of the promoter and exon 1 of the gene, and approximately 3.5 kb upstream of a previously reported autonomously replicating sequence (Sykes et al. [ 1988 ] Mol. Gen. Genet. 212:301–309). J. Cell. Biochem. 85: 346–356, 2002.

A Late Origin of DNA Replication in the Trinucleotide Repeat Region of the Human FMR2 Gene

We confirmed that the replication of the fragile-X E site (FRAXE) in human chromosomal band Xq28 occurs at six hours into the eight-hour S phase of normal human fibroblasts. In this late-replicating region, we mapped an origin of DNA replication within the promoter of FMR2. This origin is coincident with CpG islands, the trinucleotide repeat, and exon 1 of the FMR2 gene. Identification of this origin may aid in the investigation of the mechanism of trinucleotide repeat expansion and its effect on FMR2 expression. In addition, knowledge of the chromosomal locations and sequence characteristics of both early and late origins of DNA replication, such as the one described in this report, will facilitate studies of the molecular determinants of the time of activation of different origins of replication and allow us to refine our insights concerning origin inactivation in response to the DNA damage-induced intra-S checkpoint.

Transitions in replication timing in a 340 kb region of human chromosomal R‐Band 1p36.1

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase (Cohen et al. [1998]: Exp Cell Res. 245:321–329). In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1–2 h later. Using a quantitative PCR‐based assay to measure sequence abundance in size‐fractionated (900–1,700 nt) nascent DNA (Giacca et al. [1994]: Proc Natl Acad Sci USA. 91:7119), we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.

Construction of a cosmid library of DNA replicated early in the S phase of normal human fibroblasts.

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by release from confluence arrest and incubation in the presence of aphidicolin. Bromodeoxyuridine (BrdUrd) was added to aphidicolin‐containing medium to label DNA replicated as cells entered S phase. Nuclear DNA was partially digested with Sau 3AI, and hybrid density DNA was separated in CsCl gradients. The purified early‐replicating DNA was cloned into sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High‐density colony hybridization filters (1,536 clones/filter) were prepared from a set of replicas of the original plates. Bacteria remaining in the wells of replica plates were combined, mixed with freezing medium, and stored at −80°C. These pooled stocks were analyzed by polymerase chain reaction to determine the presence of specific sequences in the library. Hybridization of high‐density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96‐well plates. In testing the library for the presence of 14 known early‐replicating genes, we found sequences at or near 5 of them: APRT, β‐actin, β‐tubulin, c‐myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication. J. Cell. Biochem. 78:509–517, 2000.

DNA damage checkpoint responses in the S phase of synchronized diploid human fibroblasts.

We investigated the hypothesis that the strength of the activation of the intra‐S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m−2 UVC in early, mid‐ and late‐S phase. The endpoints measured were the following: (1) radio‐resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC‐induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10‐fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra‐S checkpoint activated by UVC‐induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.