Helmut Blum

LBR and Lamin A/C Sequentially Tether Peripheral Heterochromatin and Inversely Regulate Differentiation

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs

With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%--5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.

Monozygotic Twin Model Reveals Novel Embryo-Induced Transcriptome Changes of Bovine Endometrium in the Preattachment Period

Abstract Initiation and maintenance of pregnancy are critically dependent on an intact embryo-maternal communication in the preimplantation period. To get new insights into molecular mechanisms underlying this complex dialog, a holistic transcriptome study of endometrium samples from Day 18 pregnant vs. nonpregnant twin cows was performed. This genetically defined model system facilitated the identification of specific conceptus-induced changes of the endometrium transcriptome. Using a combination of subtracted cDNA libraries and cDNA array hybridization, 87 different genes were identified as upregulated in pregnant animals. Almost one half of these genes are known to be stimulated by type I interferons. For the ISG15ylation system, which is assumed to play an important role in interferon tau (IFNT) signaling, mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found at increased levels in addition to ISG15 and UBE1L. These results were further substantiated by colocalization of these mRNAs in the endometrium of pregnant animals shown by in situ hybridization. A functional classification of the identified genes revealed several different biological processes involved in the preparation of the endometrium for the attachment and implantation of the embryo. Specifically, elevated transcript levels were found for genes involved in modulation of the maternal immune system, genes relevant for cell adhesion, and for remodeling of the endometrium. This first systematic study of maternal transcriptome changes in response to the presence of an embryo on Day 18 of pregnancy in cattle is an important step toward deciphering the embryo-maternal dialog using a systems biology approach.

The endometrium responds differently to cloned versus fertilized embryos

Although somatic cell nuclear transfer (SCNT) cloning is more efficient in cattle than in any other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo–maternal interactions in the peri-implantation period. Therefore, we evaluated the response of the endometrium to SCNT embryos (produced from 7 different fetal fibroblast cell lines) as compared with embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (day 7) and were transferred to corresponding recipients, which were slaughtered at day 18 of pregnancy. The mRNA profiles of endometrium samples were obtained using a custom cDNA microarray enriched for transcripts differentially expressed in the endometrium and/or oviduct epithelium during the estrous cycle and/or early pregnancy. Overall, the variation in mRNA profiles was greater in the SCNT group than in the IVF group. Furthermore, 58 transcripts were differentially abundant in endometria from SCNT and IVF pregnancies. Prominent examples are orphan nuclear receptor COUP-TFII and connexin 43, both known to play important roles in uterine receptivity and conceptus placentation. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo–maternal communication that develops during the peri-implantation period. Endometrium transcriptome profiles may serve as a tool to evaluate SCNT embryos for their ability to establish pregnancy and develop a functional placenta.

Comparative Kinetics of Escherichia coli- and Staphylococcus aureus-Specific Activation of Key Immune Pathways in Mammary Epithelial Cells Demonstrates That S. aureus Elicits a Delayed Response Dominated by Interleukin-6 (IL-6) but Not by IL-1A or Tumor Necrosis Factor Alpha

ABSTRACT Infections of the udder by Escherichia coli very often elicit acute inflammation, while Staphylococcus aureus infections tend to cause mild, subclinical inflammation and persistent infections. The molecular causes underlying the different disease patterns are poorly understood. We therefore profiled the kinetics and extents of global changes in the transcriptome of primary bovine mammary epithelial cells (MEC) after challenging them with heat-inactivated preparations of E. coli or S. aureus pathogens. E. coli swiftly and strongly induced an expression of cytokines and bactericidal factors. S. aureus elicited a retarded response and failed to quickly induce an expression of bactericidal factors. Both pathogens induced similar patterns of chemokines for cell recruitment into the udder, but E. coli stimulated their synthesis much faster and stronger. The genes that are exclusively and most strongly upregulated by E. coli may be clustered into a regulatory network with tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) in a central position. In contrast, the expression of these master cytokines is barely regulated by S. aureus. Both pathogens quickly trigger an enhanced expression of IL-6. This is still possible after completely abrogating MyD88-dependent Toll-like receptor (TLR) signaling in MEC. The E. coli-specific strong induction of TNF-α and IL-1 expression may be causative for the severe inflammatory symptoms of animals suffering from E. coli mastitis, while the avoidance to quickly induce the synthesis of bactericidal factors may support the persistent survival of S. aureus within the udder. We suggest that S. aureus subverts the MyD88-dependent activation of immune gene expression in MEC.

Whole-exome sequencing in adult ETP-ALL reveals a high rate of DNMT3A mutations

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a high-risk subgroup of T-lineage ALL characterized by specific stem cell and myeloid features. In adult ETP-ALL, no comprehensive studies on the genetic background have been performed to elucidate molecular lesions of this distinct subgroup. We performed whole-exome sequencing of 5 paired ETP-ALL samples. In addition to mutations in genes known to be involved in leukemogenesis (ETV6, NOTCH1, JAK1, and NF1), we identified novel recurrent mutations in FAT1 (25%), FAT3 (20%), DNM2 (35%), and genes associated with epigenetic regulation (MLL2, BMI1, and DNMT3A). Importantly, we verified the high rate of DNMT3A mutations (16%) in a larger cohort of adult patients with ETP-ALL (10/68). Mutations in epigenetic regulators support clinical trials, including epigenetic-orientated therapies, for this high-risk subgroup. Interestingly, more than 60% of adult patients with ETP-ALL harbor at least a single genetic lesion in DNMT3A, FLT3, or NOTCH1 that may allow use of targeted therapies.

Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle.

During the oestrous cycle, the bovine endometrium exhibits characteristic morphological and functional changes, which are mainly induced by progesterone (P(4)), oestrogens and oxytocin. We studied the response of the endometrium to this changing hormonal environment at the transcriptome level using a custom-made cDNA microarray. Endometrium samples were recovered from Simmental heifers on days 0 (oestrus), 3.5 (metoestrus), 12 (dioestrus) and 18. The latter group was divided into animals with high (late dioestrus) and low P(4) levels (preoestrus). Significance analysis of microarrays revealed 269 genes exhibiting significant changes in their transcript levels during the oestrous cycle in distinct temporal patterns. Two major types of expression profiles were observed, which showed the highest mRNA levels during the oestrus phase or the highest levels during the luteal phase respectively. A minor group of genes exhibited the highest mRNA levels on day 3.5. Gene ontology (GO) analyses revealed GO categories related to extracellular matrix remodelling, transport, and cell growth and morphogenesis enriched at oestrus, whereas immune response and particular metabolic pathways were overrepresented at dioestrus. Generation of gene interaction networks uncovered the genes possibly involved in endometrial remodelling (e.g. collagen genes, TNC, SPARC, MMP2, MEP1B, TIMP1, TIMP2, HTRA1), regulation of angiogenesis (e.g. ANGPTL2, TEK, NPY, AGT, EPAS1, KLF5 ), regulation of invasive growth (e.g. PCSK5, tight junction proteins, GRP, LGALS1, ANXA2, NOV, PLAT, MET, TDGF1, CST6, ITGB4), cell adhesion (e.g. MUC16, LGALS3BP) and embryo feeding (e.g. SLC1A1, SLC11A2, SLC16A1, SEPP1, ENPP1). Localisation of mRNA expression in the endometrium was analysed for CLDN4, CLDN10, TJP1, PCSK5, MAGED1, and LGALS1.

Escherichia coli infection induces distinct local and systemic transcriptome responses in the mammary gland

BackgroundColiform bacteria are the most common etiologic agents in severe mastitis of cows. Escherichia coli infections are mostly restricted to a single udder quarter whereas neighboring quarters stay clinically inapparent, implicating the presence of a systemic defense reaction. To address its underlying mechanism, we performed a transcriptome study of mammary tissue from udder quarters inoculated with E. coli (6 h and 24 h post infection), from neighboring quarters of the same animals, and from untreated control animals.ResultsAfter 6 h 13 probe sets of differentially expressed genes (DEG) were detected in infected quarters versus control animals. Eighteen hours later 2154 and 476 DEG were found in infected and in neighboring quarters vs. control animals. Cluster analysis revealed DEG found only in infected quarters (local response) and DEG detected in both infected and neighboring quarters (systemic response). The first group includes genes mainly involved in immune response and inflammation, while the systemic reaction comprises antigen processing and presentation, cytokines, protein degradation and apoptosis. Enhanced expression of antimicrobial genes (S100A8, S100A9, S100A12, CXCL2, GNLY), acute phase genes (LBP, SAA3, CP, BF, C6, C4BPA, IF), and indicators of oxidative stress (GPX3, MT1A, MT2A, SOD2) point to an active defense reaction in infected and neighboring healthy quarters. Its early onset is indicated by increased transcription of NFIL3 at 6 h. NFIL3 is a predicted regulator of many genes of the systemic response at 24 h. The significance of our transcriptome study was evidenced by some recent findings with candidate gene based approaches.ConclusionsThe discovery and holistic analysis of an extensive systemic reaction in the mammary gland significantly expands the knowledge of host-pathogen interactions in mastitis which may be relevant for the development of novel therapies and for genetic selection towards mastitis resistance.

Fine mapping of genome activation in bovine embryos by RNA sequencing

Significance Gene expression profiling is widely used to get insight into mechanisms of early embryonic development and to characterize embryos generated by various techniques or exposed to different culture conditions. Transcripts in early embryos may be of maternal or embryonic origin, which is difficult to distinguish by conventional techniques. RNA sequencing in bovine oocytes and embryos facilitated mapping of the onset of embryonic expression for almost 7,400 genes. The timing of embryonic gen(om)e activation offers an additional level of information for embryo biosystems research and for detecting disturbances of early development due to genetic, epigenetic, and environmental factors. During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 103 different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation [i.e., (i) 59%, (ii) 42%, and (iii) 58%] was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.

Transcriptome Studies of Bovine Endometrium Reveal Molecular Profiles Characteristic for Specific Stages of Estrous Cycle and Early Pregnancy

The endometrium undergoes marked functional changes during estrous cycle and pregnancy. As the adjacent environment of the conceptus, it represents the maternal interface for embryo-maternal communication, which is essential to maintain pregnancy. Transcriptome studies provide the unique opportunity to assess molecular profiles changing in response to endocrine or metabolic stimuli or to embryonic pregnancy recognition signals. Here we review the current state of transcriptome profiling techniques and the results of a series of transciptome studies comparing bovine endometrium samples during the estrous cycle or endometrium samples from pregnant vs. non-pregnant animals. These studies revealed specific mRNA profiles which are characteristic for the functional status of the endometrium. Transcriptome studies of endometrial samples recovered during the pre-attachment period identified many interferon-stimulated genes, genes that are possibly involved in embryo-maternal immune modulation ( C1S, C1R, C4, SERPING1, UTMP, CD81, IFITM1, BST2), as well as genes affecting cell adhesion ( AGRN, CD81, LGALS3BP, LGALS9, GPLD1, MFGE8, and TGM2) and remodeling of the endometrium ( CLDN4, MEP1B, LGMN, MMP19, TIMP2, TGM2, MET, and EPSTI1). The results of these transcriptome studies were compared to those of similar microarray analyses in human, mouse and Rhesus monkey to identify similarities in endometrial biology between mammalian species and species-specific differences. Future studies will cover dynamic transcriptome changes between different stages of early pregnancy, the relationship between metabolic problems in dairy cows and the functionality of reproductive tissues as well as endometrium transcriptome profiles in recipients of somatic cell nuclear transfer embryos.