Hinco J Gierman

Abstract: Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1β (HP1β) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1β protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the chromatin pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Abstract: In conducting genome-wide association studies (GWAS), analytic approaches leveraging biologic information may further understanding of the pathophysiology of clinical traits. To discover novel associations with estimated glomerular filtration rate (eGFR), a measure of kidney function, we developed a strategy for integrating prior biological knowledge into existing GWAS data for eGFR from the CKDGen Consortium. Our strategy focuses on SNPs in genes that are connected by functional evidence, determined by literature mining and gene ontology hierarchies, to genes near previously validated eGFR associations. It then requires association thresholds consistent with multiple testing, and finally evaluates novel candidates by independent replication. Among samples of European ancestry, we identified a genome-wide significant SNP in FBXL20 (p=5.6x10(-9)) in meta-analysis of all available data, and additional SNPs at the INHBC, LRP2, PLEKHA1, SLC3A2, and SLC7A6 genes meeting multiple-testing corrected significance for replication and overall p-values of 4.5x10(-4)-2.2x10(-7). Neither the novel PLEKHA1 nor FBXL20 associations, both further supported by association with eGFR among African Americans and with transcript abundance, would have been implicated by eGFR candidate gene approaches. LRP2, encoding the megalin receptor, was identified through connection to the previously known eGFR gene DAB2 and extends understanding of the megalin system in kidney function. These findings highlight integration of existing genome-wide association data with independent biological knowledge to uncover novel candidate eGFR associations, including candidates lacking known connections to kidney-specific pathways. The strategy may also be applicable to other clinical phenotypes, although more testing will be needed to assess its potential for discovery in general.

Abstract: Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD.

Abstract: Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10-30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5- to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin-chromatin interactions and determine the 3-dimensional organization of the chromatin fiber.

Abstract: Transcription factor complexes bind to regulatory sequences of genes, providing a system of individual expression regulation. Targets of distinct transcription factors usually map throughout the genome, without clustering. Nevertheless, highly and weakly expressed genes do cluster in separate chromosomal domains with an average size of 80-90 genes. We therefore asked whether, besides transcription factors, an additional level of gene expression regulation exists that acts on chromosomal domains. Here we show that identical green fluorescent protein (GFP) reporter constructs integrated at 90 different chromosomal positions obtain expression levels that correspond to the activity of the domains of integration. These domains are up to 80 genes long and can exert an eightfold effect on the expression levels of integrated genes. 3D-FISH shows that active domains of integration have a more open chromatin structure than integration domains with weak activity. These results reveal a novel domain-wide regulatory mechanism that, together with transcription factors, exerts a dual control over gene transcription.

Abstract: The three-dimensional (3D) organization of the chromosomal fiber in the human interphase nucleus is an important but poorly understood aspect of gene regulation. Here we quantitatively analyze and compare the 3D structures of two types of genomic domains as defined by the human transcriptome map. While ridges are gene dense and show high expression levels, antiridges, on the other hand, are gene poor and carry genes that are expressed at low levels. We show that ridges are in general less condensed, more irregularly shaped, and located more closely to the nuclear center than antiridges. Six human cell lines that display different gene expression patterns and karyotypes share these structural parameters of chromatin. This shows that the chromatin structures of these two types of genomic domains are largely independent of tissue-specific variations in gene expression and differentiation state. Moreover, we show that there is remarkably little intermingling of chromatin from different parts of the same chromosome in a chromosome territory, neither from adjacent nor from distant parts. This suggests that the chromosomal fiber has a compact structure that sterically suppresses intermingling. Together, our results reveal novel general aspects of 3D chromosome architecture that are related to genome structure and function.

Abstract: Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.

Abstract: The expression of genes in the human genome is regulated by transcription factor complexes. These complexes bind to regulatory sequences, usually in the promoter region of a gene. The concentration or composition of these complexes determines the amount of messenger ribonucleic acid (mRNA) that is produced. This system of individual gene regulation in principle allows genes to be randomly positioned throughout the genome and this was assumed to be the case until recently.

Abstract: We look for organic compounds with novel structure as well as potential antimicrobial and antioxidative activity. We have chosen to work with phytoopathogenic fungi, because of their capability of transforming complex compounds such as the terpenoids found in plants. As substrate, (-)-Ambroxide (1) was chosen since there is little bibliographical data on its biotransformation (and it has a formal structural relationship to the well known cytotoxic Sclareolide). The biotransformation was carried out in a two stage protocol. Biotransformation was achieved after 14 days at 25ªC. 5 transformed compounds were identified and purified and analysed by means of spectroscopic methods. The major product has been identified as 3�, 6� -dihydroxyambroxide (2). This is the first report of biotransformation of substrate 1 by Wilsonomyces carpophilus. We can see that the fungus is capable of stereospecific hydroxylation of substrate 1 in the 3 and 6 position. The structural properties of the other products will be elucidated and antimicrobial and antioxidative activity will be established. Using fitopathogens proves to be a successful approach for the transformation of complex compounds and might lead to the discovery of novel compounds with antimicrobial or antioxidatve capacities.

Abstract: Chromosomes are the long DNA molecules that carry the genetic code of our genes. Each gene encodes a protein, but also contains the information that controls the activity of that gene. In this thesis, we find that chromosomal domains with many active genes (so-called 'Ridges'), also control gene activity. Our results imply that Ridges facilitate the high activity of important genes. Ridges might thus provide an alternative mechanism for changes in gene activity during human evolution.