Molecular Insights into Development in Humans

Studies in Normal Development and Birth Defects

Moyra Smith

• Editore: WSPC
• Anno: 2014
• ISBN:
  9789814630603
• Argomento: Medicine
• Categoria: Biochemistry in Medicine

The aim and scope of this book is to review current information on human development and processes of differentiation that have benefited from breakthrough analyses in stem cell biology, elucidation of genome and gene architecture and aspects of regulation of gene expression, analysis of signaling systems and transcription factor actions.

Insights into actions of specific genes and their roles in development have been gathered through studies in patients with specific birth defects, including congenital malformations, metabolic defects and functional impairments.

The book is organized into three sections, the first dealing with aspects of genomics, gene structure and regulation, analysis of signaling and function of specific organelles. The second section deals with molecular aspects of development of specific organs and structures such as, bone, face, brain, heart, liver, pancreas, kidney. The last section deals with specific malformations and tumors that provide insight into regulation of growth. Environmental factors that impact growth and development are also covered.

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Contents:

• Genomes, Genes, Structure and Function
• Epigenetics
• Signaling Systems
• Pluripotency to Differentiation
• Development and Growth Abnormalities
• Metabolism and Organelles
• Brain Development
• Cranio–Facial Development and Defects
• Molecular Aspects of Heart Development
• Vasculogenesis Malformations and Hematopoiesis
• Abdominal Wall and Gastro-Intestinal Tract
• Lung and Diaphragm Development
• Liver and Pancreas Development
• Bone and Extra-Cellular Matrix
• Kidney and Urinary Tract Development, Malformations in Human Genes
• Sex Determination Differentiation and Endocrine Glands

Readership: Graduate students, physicians, geneticists, genetic counsellors and researchers in development biology.
Key Features:

• The concepts of receptor theory and hierarchical levels of pharmacological analysis are integrated and emphasised throughout the book
• Step-by-step instructions are given for the different types of pharmacological analyses

CHAPTER 1

GENOMES, GENES, STRUCTURE AND FUNCTION

Molecular embryology involves analysis of gene expression and delineation of the key gene products involved in determining differentiation at the cellular and tissue level.

Overlapping Layers within Genome Architecture

Overlapping layers within the genome architecture need to be taken into account in analyzing gene expression, these include:

(a) The linear sequence of DNA and linear gene structure;

(b) The embedding of DNA in histone rich chromatin that undergoes modifications that impact gene expression;

(c) The arrangement of DNA and chromatin within the nucleus at different stages of the cell cycle.

During metaphase distinct chromosomes can be identified. More precise identification of each of the human chromosomes was initially achieved through development of specific histological staining techniques that revealed banding patterns on chromosomes (Caspersson et al., 1970; Seabright, 1972). Molecular cytogenetics began in 1977 when labeled DNA probes were hybridized to chromosomes.

Analysis of folding and looping of DNA and chromatin in interphase nuclei and of three-dimensional structure is currently being actively analyzed. There is evidence that gene regulation is influenced by looping of chromatin and DNA and by dynamic changes in the positioning of loops on which specific genes and regulators are positioned. Fluorescence in-situ hybridization (FISH) techniques provided means to examine the location of specific gene targets within the nucleus. FISH studies initially provided evidence that specific chromosomes have preferred positions in the nucleus. The arrangement differed in different cell types (Bickmore, 2013).

Molecular Organization of Eukaryotic Genes

The molecular organization of eukaryotic genes, including human genes has been actively ongoing since the late 1970s when it became clear that these genes contain coding segments (exons) interspersed with noncoding segments introns (Leder, 1978). Analyses revealed that transcription is initiated from the 5 prime (5′) end of the gene from a site adjacent to promoter sequences that are located further 5′ (upstream) of the transcription initiation site. The promoter sequences are not transcribed but comprise elements that facilitate transcription: specific regions within the promoter bind polymerases essential for transcription.

Detailed molecular studies revealed that the primary mRNA transcript undergoes cleavage at specific points and subsequent rejoining and that this splicing process removes introns. The precise location of splicing sometimes varied depending on the nucleotide sequences present at specific positions. Specific nucleotide sequences at the 3′ end of the transcript directs binding of an endonuclease that lead to cleavage at the 3′ end and this cleavage site then polyadenylated.

Developmental Control of Gene Expression

Development is characterized by temporal and spatial differences in gene expression. Development requires the integrative action of gene promoters and cis-regulatory elements including those that lie close to promoters and those that lie at some distance from transcription start sites. Important cis-acting elements that impact gene expression include enhancers, silencers, insulators and transcription factor binding sites.

In this section, I review information on promoters, on cis-regulatory elements including enhancers and transcription factors and their binding sites. Following this aspects of transcription, alternative splicing, alternate generation of 3′ end sequences and aspects of translation are then reviewed.

Pal et al. (2011) emphasized that analysis of transcription and its regulation is essential for deciphering cell and tissue specific gene functions. Alternative transcription initiation and alternative transcription termination give rise to alternative mRNA transcripts and these may undergo alternative splicing and then give rise to alternate protein isoforms.

Promoters

Promoters are not transcribed; they contain sequence elements that enhance transcription capability. Promoters include core domains and regulatory domains. Key sequence elements within the promoters include the polymerase II binding site and transcription binding sites. Protein coding genes are transcribed by RNA polymerase II and core elements included in these genes include TATA boxes (sequence rich in thymine and adenine), CpG islands (repeats of cytosine and guanine) are often present in the promoter regions. Chromatin modifications are key in determining transcription initiation and are discussed in the subsequent chapter on epigenetics. TATA boxes are present in promoters of approximately 24% of human genes. Key to transcription of TATA box-containing promoters is a TATA box binding protein (TBP). This protein binds to the TATA box and acts to properly position the RNA polymerase II. It also serves as a scaffold for the assembly of transcription factors.

Alternate promoter use

Different stages of development and different cellular and tissue conditions may require use of different promoters that are differentially regulated. Furthermore sequence differences in promoters lead to binding of different transcription factors.

Factors that determine the use of alternate promoters include DNA sequences in regulatory regions and histone modifications. Pal et al. (2011) reported that 50% of the multi-transcript genes they studied used multiple promoters. They also determined that genes which used multiple promoters also exhibited alternative transcription termination and alternative splicing. They carried out mRNA sequencing and epigenetic analysis to develop an inventory of transcript variants that occur in development of the cerebellum in the mouse. Their studies revealed extensive changes in the expression of transcript variants of a number of different genes during differentiation of the granular cells of the cerebellum.

Examples of genes with multiple promoters

The uridine diphosphate glucuronosyl transferase 1A (UGT1A) gene on human chromosome 2q37 has 13 alternative promoters. The BDNF gene that encodes “brain derived neurotrophic factor” is expressed from nine different promoters. The gene gives rise to multiple different transcripts that result from use of different promoters, different transcription start sites and from alternative splicing of exons. Neuronal activity impacts BDNF transcription and splicing (Autry and Monteggia, 2012).

Sakata et al. (2009) reported that BDNF promoter IV plays a particularly important role in BDNF transcription. They reported that absence of that promoter in mice led to deficits in specific neurotransmitter functions in gamma amino butyric acid (GABA) ergic interneurons in the prefrontal cortex.

Methods to analyze alternate promoters of a specific gene:CAP sequencing

Messenger RNA contains a 5′ CAP sequence in which 7-methylguanosine is linked to the first transcribed nucleotide in a phosphodiesterase bond. The CAP synthesizing complex is associated with RNA polymerase. CAGE sequencing is a sequencing method that involves biotinylation of this 5′ 7-methylguanosine and then selection through streptavidin binding. This capture enables sequencing of 5′ mRNA adjacent to the 5′ CAP. CAGE sequencing has revealed that most genes have multiple promoters and there is frequent tissue specific or developmental stage specific promoter usage. Faulkner et al. (2009) reported that repetitive DNA and retro-transposons (ancient sequences that can amplify themselves and are mobile in the genome) are often associated with the 5′ region of protein coding genes and may serve as alternate promoters. They proposed that retro-transposon transcription has a key influence on the transcriptional output of the mammalian genome.

Promoters have a 3′ splice donor but lack a 5′ splice sequence. Each different promoter can then bind to the downstream exon of a gene through binding to the splice acceptor adjacent to that exon.

Transcription

Transcription factors are key constituents of gene expression regulatory systems and are central elements in determination of differentiation and development. Two distinct domains that occur within transcription factor proteins include a DNA binding domain that permits binding to sequence elements in DNA and secondly an activator domain that directly impacts transcription. In some cases the transcription factor interacts with a coactivator domain. The repertoire of transcription factors includes general or basal transcription factors that facilitate transcription and specific transcription factors that bind only to specific DNA sequences.

Transcription initiation

Control of transcription initiation is essential to development. Distal and proximal enhancers and promoters are involved in this process. Transcription start sites can be identified through analysis of sequence at the 5′ end of full-length cDNAs (complementary DNA segments sequenced from messenger RNA). Kawaji et al. (2006) reported that for a specific gene, transcription start site selection varied in different tissues and that most genes do not have a specific single transcription start site. They determined that for a specific gene, alternative start sites were present and these were spread across the 5′ gene region. Furthermore for a specific gene transcription, start site selection varied in different tissues.

Recent studies have revealed that transcription factors may bind at the transcription start site or downstream or upstream of that site. The transcription machinery that assembles at the promoter includes 27 polypeptides including general transcription factors.

Enhancers contain specific DNA sequence elements to which transcription factors bind. However the low sequence specificity of the 6–12 nucleotide elements that bind transcription factors results in the potential for a specific sequence element to bind different transcription factors. It is therefore possible that at different stages of development different transcription factors may bind to a specific enhancer element. Furthermore it seems likely that different combinations of transcription factors may bind to a specific enhancer site.

Pioneer transcription factors

Spitz and Furlong (2012) reviewed evidence for the existence and function of pioneer transcription factors. These bind to specific genomic sequence sites and enhancer sequences and lead to alterations in nucleosome positioning in adjacent genomic regions. The pioneer transcription factors do not necessarily activate the enhancers to which they bind. The pioneer transcription factors may subsequently be replaced by other transcription factors. Pioneer transcription factors may also serve to protect enhancer sites from methylation. Examples of transcription factors that may have pioneer function include MyoD and PAX5.

Spitz and Furlong (2012) emphasized that expression of a specific gene is dependent upon enhancers, available promoter elements and the three-dimensional arrangement of genome segments.

An important consideration is whether structural chromosome changes; e.g., duplication, deletions, translocations and inversions alter the three-dimensional chromosome organization and the potential for promoter enhancer interactions.

Transcription factors and pluripotency of cells

The field of transcription factor research took a great leap forward with the discovery of the key role of transcription factors in converting somatic cells, such as fibroblasts, to pluripotent stem cells. Stem cells and induction of pluripotency and differentiation will be discussed in a subsequent chapter.

Function and expression of human transcription factors

In a review of human transcription factors, Vaquerizas et al. (2009) presented data on 1,391 manually curated sequence specific transcription factors. Complete genome sequence analysis of DNA elements that bind to transcription factors have led to the creation of a number of different databases with inventories of transcription factors. Classifications of transcription factors are based on DNA binding characteristics, on protein domains and structural homologies and on the basis of the biological processes in which they participate. Vaquerizas et al. (2009) noted that in some cases the structural characteristics of a transcription factor provide some insight into its biological function, e.g., homeodomain transcription factors are often involved in developmental processes. Classifications are sometimes based on the tissue or tissue in which the transcription factor is expressed. It is important to note that transcription factors undergo extensive protein–protein interactions and in some cases combinations of transcription factors determine regulation.

Vaquerizas et al. (2009) classified transcription factors into 23 families and added a 24 “other” category for undefined transcription factors. Zinc finger transcription factors constituted the most predominant class, approximately 680 of 700, followed by homeodomain factors approximately 250 of 700 and the helix-loop-helix transcription factors 80 of 700. They also classified transcription factors according to biological function based on literature reports. Of 741 factors thus analyzed 263 were involved in developmental processes, 221 in cellular processes, 109 in metabolic processes, 66 in responses to stimuli, 30 in immune processes, 28 in reproductive processes and 24 in localization.

Vaquerizas et al. (2009) reported that results of expression studies revealed that approximately one third of transcription factors were expressed primarily in one tissue. These included the heart specific transcription factor NKX2-1, and the fetal brain e...