Molecular Regulation and Signaling (Langman's Part 1) PDF

Introduction to to Molecular Molecular Regulation and Signaling Molecular Molecular biology biology has has opened opened the the doors to new Histone complex ways to ways...

Introduction to to Molecular Molecular Regulation and Signaling Molecular Molecular biology biology has has opened opened the the doors to new Histone complex ways to ways study embryology to study embryology and and to enhance our to enhance DNA understanding of understanding ofnormal normal andand abnormal abnormal develop- develop- Sequencing the human genome, together ment. Sequencing with creating techniques to to investigate investigate gene reg- many levels ulation at many levels of complexity, complexity, has taken - -Nucleosome Nucleosome embryology embryology to to the the next level. level. Thus, Thus, from from the anatomical anatomical to the biochemical biochemical to the molecular H1 level, the level, the story story of ofembryology embryology has has progressed, progressed, histones Linker and each chapter has enhanced our our knowledge. knowledge. DNA Embryonic development Embryonic development isis directed directed by ge- ge- nomes nomes that contain contain allall of of the the information information re- re- FIGURE 1.1 FIGURE 1.1Drawing Drawing showing nucleosomes that showing nucleosomes quired to make make an an individual. individual. TheThe information information basic unit form the basic unit of of chromatin. chromatin. Each Each nucleosome nucleosome is is encoded encoded in DNA DNA in in sequences sequences called called genes genes consists consists of anan octamer of histone proteins and ap- ap— proximately proximately 140140 base base pairs pairs of of DNA. DNA. Nucleosomes Nucleosomes proteins regulate that code for proteins. In turn, proteins regulate are are joined by linker joined into clusters by linker DNA DNA and and other his- his— expression of other genes and act act as as signal mole- tone proteins. proteins. cules to orchestrate development. development. There are approximately 23,000 There 23,000 genes genes inin the human genome, genome, which represents only only one-fifth one-fifth of the number (100,000) (100,000) predicted prior to com- and andits and its basic basic unit of structure is the the nucleosome nucleosome pletion of the Human GenomeGenome Project. Project. Because Because (Fig. 1.1). (Fig. Each nucleosome 1.1). Each nucleosome isis composed composed of an of various various levels levels ofof regulation, regulation, however, however, the the octamer of histone proteins proteins and and approximately approximately number of of proteins derived derived from thesethese genes genes is is 140 base 140 base pairs pairs of DNA. DNA. Nucleosomes Nucleosomes themselves closer to the original predicted number number of ofgenes. genes. are joined into clusters clusters by binding of of DNA DNA ex-ex- What has been disproved disproved is is the one gene-one gene—one between nucleosomes isting between nucleosomes (linker DNA) with protein hypothesis. protein Thus, through hypothesis. Thus, through a variety variety of other histone histone proteins proteins (H1 (H1 histones; histones; Fig. Fig. 1.1). 1.1). mechanisms, aa single mechanisms, single gene may give rise to many Nucleosomes keep the DNA tightly coiled, such proteins. that itit cannot that cannot be be transcribed. transcribed. In In this this inactive inactive Gene expression Gene expression can be regulated regulated at at several several state, state, chromatin chromatin appears appears as as beads beads of of nucleo- nucleo- levels: (1) levels: Different genes (1) Different genes may transcribed, may be transcribed, somes somes on a string of DNADNA and is is referred to asas (2) DNA (2) from aa gene DNA transcribed from gene may may bebe selec- selec- heterochromatin. heterochromatin. For transcription transcription to occur, occur, tively processed tively processed to regulate which RNAs RNAs reach this this DNA DNA must must be be uncoiled uncoiled from from the beads. beads. In the cytoplasm the cytoplasm toto become become messenger messenger RNAsRNAs this this uncoiled uncoiled state, state, chromatin chromatin isis referred to as referred to as (mRNAs), (3) (mRNAs), (3) mRNAs may be selectively trans- euchromatin. euchromatin. lated, lated, and (4) (4) proteins made made from from the the mRNAs mRNAs Genes reside Genes reside within within the the DNA DNA strand strand and and may be differentially modified. modified. contain contain regions called exons, regions called exons, which which cancan bebe translated into translated into proteins, proteins, and and introns, which which are are interspersed between exons interspersed between exons and and which which areare not IGENE GENETRANSCRIPTION TRANSCRIPTION transcribed into proteins (Fig. (Fig. 1.2). 1.2). In addition Genes are Genes are contained contained in a complex complex of DNA DNA and to exons and introns, aa typical typical gene includes the proteins proteins (mostly (mostly histones) histones) called called chromatin, chromatin, following: aa promoter following: promoter region that binds binds RNA RNA 33  4 Partl| - General Part General Embryology Embryology Promoter Promoter region region Exon 1 Exon 1 Intron Intron 1 1 Exon Exon 22 Intron Intron 22 Exon Exon 33 Intron Intron 3 Exon Exon 4 I I I I I 3' region I untranslated region 3' untranslated 3 pi Slim 1 Jpn J 1 ill __.*.l.ii\ TATA TATA Translation Translation Enhancer Enhancer Translation Translation Transcription Transcription box box initiation initiation sequence sequence termination II termination termination termination codon codon PoAA Slte Poly site addition site addition site FIGURE 1.2 FIGURE 1.2 Drawing Drawing of of aa “typical” "typical" gene gene showing showing the region containing the promoter region containing the TATA TATA box; box; exons exons that that contain DNADNA sequences sequencesthat that are aretranslated translated into into proteins; proteins; introns; introns; the the transcription transcription initiation site; the transla— transla- initiation site tion initiation site that that designates designates the the code code for for the first amino acid rst amino acid in in a a protein; protein; and and the the 3’ region 3' untranslated region that includes that includes the the poly poly AA addition addition site site that that participates participates inin stabilizing stabilizing the the mRNA, mRNA, allows allows itit to to exit the nucleus, nucleus, and permits and permits its its translation into a a protein. protein. polymerase for the initiation initiation of transcription; transcription; a activate gene activate expression by gene expression causing the DNA by causing DNA transcription transcription initiation initiationsite; site; a translation translation initi- nucleosome complex nucleosome complex to unwind, by releasing the ation site ation site to to designate designate the first first amino acid in the polymerase polymerase so so that that it can can transcribe transcribe thethe DNA DNA protein; aa translation protein; translation termination termination codon;codon; andand a template, and by preventing template, preventing new new nucleosomes nucleosomes 3' untranslated untranslated regionregion that includes includes a sequence sequence from forming. (the poly A addition site) site) that that assists assists with stabi- Enhancers are are regulatory regulatory elements elements of of DNA DNA lizing the mRNA, allows it to exit exit the the nucleus, nucleus, and that activate activate utilization promoters to control utilization of promoters permits itit to be translated translated into into protein protein (Fig. (Fig. 1.2). 1.2). their their efficiency efficiency and and the the rate rate ofof transcription transcription By convention, By convention, the 5’ 5' and the 3' 3’ regions regions ofof a gene from from the promoter. promoter. Enhancers Enhancers can can reside reside any- any- specified in are specified in relation relation to to the RNA RNA transcribed where along the DNA DNA strand and do not have to from the gene. gene. Thus, mus, DNA DNA is transcribed from reside close reside close to to a promoter. Like Like promoters, en- the 5' 5’ to to the the 3' 3’ end, end, and and the the promoter promoter region region isis hancers hancers bind transcription factors factors (through the upstream upstream from from the transcription transcription initiation initiation site site transcription transcription factor's factor’s transactivating transactivating domain) domain) (Fig. 1.2). (Fig. 1.2). The The promoter promoter region, where the the RNA RNA and are used used to to regulate regulate the the timing timing ofof aa gene's gene’s polymerase binds, usually contains the sequence expression and its its cell-specific cell-specific location. For ex- ex- TATA, and this site is called the TATA box box (Fig. (Fig. ample, separate enhancers in a gene can be used ample, 1.2). In order 1.2). In order to bind to to this this site, site, however, however, the same gene to direct the same gene to be expressed in differ- differ- polymerase requires polymerase requires additional proteins proteins called called Hie PAX6 ent tissues. The PAX6transcription transcription factor, which transcription factorsfactors (Fig. (Fig. 1.3). 1.3). Transcription Transcription participates participates in in pancreas, pancreas, eye,eye, and and neural tubetube factors also have factors also have a specific specific DNA-binding DNA-binding do- development, contains three separate enhancers, main plus a transactivating domain that that acti- acti- each of which regulates regulates the genesgene’s expression in vates or inhibits transcription of vates of the gene gene whose the appropriate tissue. Enhancers act act by altering promoter or enhancer it has has bound. bound. In In combi- combi- chromatin chromatin to to expose expose the promoter promoteror or by by facilitat- nation with nation with other other proteins, proteins, transcription factorsfactors ing binding ofof the RNA polymerase. Sometimes, I RNA polymerase IlII\ RNA polymerase RNA polymerase II\ I| DNA DNA TATA Ihhhhhh “ulfnwl Transcription Transcription RNA transcript factor protein factor protein initiation site initiation site complex complex FIGURE 1.3 FIGURE 1.3 Drawing Drawingshowing showing binding binding of of RNA RNApolymerase polymeraseIIIlto to the the TATA TATAbox box site site of of the the promoter region of promoter region a gene. a gene. This This binding binding requires requires aa complex complex ofof proteins proteins plus plus an an additional additional protein protein called called aa transcription transcription factor. factor. Transcription factors have their own speci Transcription specific DNA—binding domain and function to c DNA-binding to regulate regulate gene gene expression. expression. fi fi fi fi  Chapter1- Chapter to Molecular 1 Introduction to Molecular Regulation Regulation and and Signaling Signaling 5 enhancers can inhibit enhancers can inhibit transcription transcription and and are are nucleosomesand nucleosomes andtightly tightly coiled coiled DNA DNA that that cannot cannot called called silencers. silencers. This This phenomenon phenomenon allows allows a transcribed be transcribed. transcription factor to activate transcription activate one one gene gene while while silencing another by silencing another by binding binding to different different en- en- hancers. Thus, hancers. Thus, transcription factors factors themselves themselves IOTHER OTHERREGULATORS REGULATORS OF OF GENE GENE EXPRESSION EXPRESSION have a DNA-binding domain specific specific to a region of DNA DNA plus plus aa transactivating transactivating domain domain that binds The initial The initial transcript of a gene gene is is called called nuclear nuclear to aa promoter promoter or or an an enhancer enhancer and and activates or activates or RNA RNA (nRNA) orsometimes (nRNA) or sometimes premessenger premessenger gene regulated by these elements. inhibits the gene RNA. RNA. ARNA nRNA isis longer longer than than mRNA mRNA because because it contains contains introns that are removed (spliced out) DNA Methylation DNA Represses Methylation Represses as as the the RNA nRNAmovesmovesfromfrom the the nucleus nucleus to the cy- cy- Transcription toplasm. In fact, toplasm. fact, this this splicing splicing process process provides provides Methylation Methylation of of cytosine cytosine basesbases in in the promoter a means for for cells cells to produce produce different different proteins proteins regions of genes genes represses represses transcription transcription of those from aa single from single gene. gene. ForFor example, example, byby removing removing genes. Thus, some genes. Thus, some genesgenes are are silenced silenced by thisthis different diiferent introns, introns, exons exons are are "spliced" different “spliced” in different mechanism. For mechanism. For example, example, one one of of the the XX chro- chro- patterns, a process patterns, process called called alternative splicing splicing mosomes mosomes in in each each cell cell of of aa female female is inactivated inactivated (Fig.1.4). (Fig. 1.4). The The process process is carried out by by spliceo- spliceo- (X chromosome inactivation) by (X by this this methyl- methyl- somes, somes, which which areare complexes complexes of of small small nuclear ation mechanism. mechanism. Similarly, genes in different Similarly, genes different RNAs RNAs (snRNAs) (snRNAs) and and proteins proteins that that recognize recognize types of cells cells are are repressed by methylation, such specific splicesites specific splice sitesatatthe the5’5' or or the the 3’ 3' ends ends of that muscle muscle cells cells make make muscle muscle proteins proteins (their (their nRNA. Proteins derived from the same gene the nRNA. promoter promoter DNA DNA is is mostly mostly unmethylated) unmethylated) but are are called called splicing isoforms (also (also called called splice not blood proteins (their (their DNA DNA isis highly methyl- variants oror alternative alternative splice splice forms), forms), and and these these ated). ated). In this manner, each each cell cell can maintain its afford the afford the opportunity forfor different different cells cells to use characteristic differentiated characteristic differentiated state. DNA DNA methyla- methyla- specific for that the same gene to make proteins specific tion is also responsible for also responsible for genomic genomicimprinting imprinting cell type. type. For example, example, isoforms of the WT1 WT] gene in which only aa gene gene inherited inherited from the father or have different have different functions functions in gonadal gonadal versus kid- mother is the mother is expressed, expressed, whereas the other gene ney neydevelopment. development. is silenced. silenced. Approximately 40 to 60 60 human human genes Even after Even after a protein protein is is made made (translated), (translated), are are imprinted, imprinted, and their their methylation methylation patterns patterns there may there may be be posttranslational modifications modifications are are established established during during spermatogenesis spermatogenesis and and that that affect affect its its function. For For example, some pro- example, some oogenesis. Methylation oogenesis. silences DNA Methylation silences DNA by inhib- teins have to be cleaved to become active, active, or they iting binding of transcription factors or transcription factors or by by alter- might have have to be phosphorylated. phosphorylated. Others need ing histone binding resulting in stabilizationstabilization of to combine combine with other proteins proteins or or be be released released 5' untranslated 5' untranslated Tissue-specific issue-specif 3' untranslated 3' untranslated region region Exons Exons exon (bone) xon (bont Introns Introns region region.-"-..r" "'-. l gaff-EEK l H #- “a. I HHypothetical th t' I | ypgeng'ca gene l. l I. I.I 11 ill [.ll'll | ____— “El , Proteinl :I:l’ ij’ i:.--"""~« _""'-._fi Protein I Protein man? Protein I|| (bone) 'bone' fit I Elfll] ‘Zi Protein III FIGURE 1.4 FIGURE 1.4 Drawing Drawingofof aa hypothetical hypothetical gene gene illustrating illustrating the the process processof of alternative alternative splicing splicingto to form form different different proteins from proteins from the the same same gene. gene. Spliceosomes Spliceosomes recognize recognize specific speci c sites sites on on the the initial initial transcript of nRNA nRNA from a a gene. Based gene. Based on on these these sites, sites, different are "spliced different introns are “spliced out” out" to create more than one protein from a single gene. Proteins gene. Proteins derived from the same same gene are are called called splicing splicing isoforms. isoforms. fi fi  6 Part I| - General Part General Embryology Embryology targeted to from sequestered sites or be targeted to specific specific mesenchyme from mesenchyme from the metanephric metanephric blastema blastema cell cell regions. regions. Thus, there are Thus, there are many many regulatory regulatory to produce nephrons in in the the kidney. kidney. Inductive Inductive levels for levels synthesizing and activating for synthesizing activating proteins, proteins, interactions can also interactions can also occur between between two two epi- epi- although only such that although only 23,000 23,000 genes genes exist, exist, the thelial tissues, thelial tissues, such such as as induction of the lenslens by potential number number ofof proteins that that can can be synthe- epithelium of the optic cup. cup. Although an initial sized is is probably closer closer to to five fivetimes times the the number number signal by the signal by the inducer to the responder responder initiates initiates of genes. the inductive event, event, crosstalk between the two two tissues or cell tissues cell types is is essential essential for for differentia- differentia- tion to continue continue (Fig. (Fig. 1.5, 1.5, arrows). arrows). IINDUCTION INDUCTION AND AND ORGAN ORGAN FORMATION ICELL CELLSIGNALING SIGNALING Organs are Organs are formed formed by by interactions interactions between between cells and tissues. cells tissues. Most often, often, one group of cells cells Cell-to-cell Cell-to-cell signaling signaling isis essential essential for induction, or tissues causes causes another set set of of cells cells or tissues tissues competency to respond, and for for conference of competency to change to change their fate, fate, aa process process called called induc- induc- crosstalk between inducing and responding respondingcells. cells. tion. In tion. In each each such such interaction, interaction, one one cell cell type type These These lines lines of communication communication are established by or tissue is is the inducer inducer that that produces produces aa signal, signal, paracrine interactions, interactions, whereby whereby proteins proteins syn- syn- and and one one is the the responder responder to to that that signal. signal. The The thesized by one cell diffuse diffuse over short distances capacity to respond capacity to respond to to such such aa signal signal is is called called to interact to interact with with other other cells, cells, or by by juxtacrine juxtacrine competence, and competence competence, competence requires requires activa- activa- interactions, which interactions, which do do not notinvolve involve diffusable diffusable tion of the responding responding tissue by a competence proteins. The diffusable diffusable proteins responsible for factor. Many Many inductive inductive interactions interactions occur be- be- signaling are paracrine signaling are called called paracrine paracrine factors tween epithelial and mesenchymal cells and are or growth and differentiation diiferentiation factors factors (GDFs). (GDFs). called called epithelial-mesenchymal epithelial—mesenchymal interactions interactions (Fig. 1.5). (Fig. 1.5). Epithelial cells are Epithelial cells are joined joined together together Signal Signal Transduction Pathways in tubes tubes or or sheets, sheets, whereas whereas mesenchymal mesenchymal cells cells Paracrine Signaling are fibroblastic fibroblastic in in appearance appearance and dispersed in Paracrine factors act Paracrine factors act by by signal signal transduction extracellular matrices (Fig. extracellular matrices (Fig. 1.5). 1.5). Examples Examples of either by pathways either by activating activating a pathway directly epithelial-mesenchymal interactions include epithelial—mesenchymal interactions include or by blocking blocking the activity activity of anan inhibitor of a the following: gut endoderm following: gut endoderm and surrounding pathway (inhibiting pathway (inhibiting an inhibitor, as is the case an inhibitor, case mesenchyme mesenchyme to to produce produce gut-derived gut-derived organs, organs, hedgehog signaling). with hedgehog signaling). Signal Signal transduction including including the liver and pancreas; pancreas; limb mesen- mesen- pathways include aa signaling pathways include signaling molecule molecule (the (the chyme chyme with overlying overlying ectoderm ectoderm (epithelium) (epithelium) and aa receptor ligand) and receptor(Fig. (Fig. 1.6). 1.6). The The receptor receptor to produce to produce limb limb outgrowth outgrowth and and differentia- differentia- spans the cell spans the membrane and cell membrane and has an extracel- extracel- tion; and tion; and endoderm endoderm of the ureteric ureteric bud andand Jular lular domain domain (the (the ligand-binding ligand-binding region), region), a transmembrane domain, transmembrane domain, and aa cytoplasmic cytoplasmic domain. When When aa ligand ligand binds binds its its receptor, receptor, it in- duces duces a conformational change in in the receptor Iffl“ isr: conformational change receptor Mesenchyme m H ' 'i that activates activates its its cytoplasmic cytoplasmic domain. domain. Usually, Usually, the result result of of this this activation activation is toto confer confer enzy- enzy- It sf? matic activity matic activity toto the receptor, receptor, and mostmost often, often, fififl “it. this activity this activity isis aa kinase kinase that can can phosphory- phosphory- Epithelium Epithelium late other proteins using ATP as as aa substrate. substrate. In turn, phosphorylation phosphorylation activates activates these proteins proteins to phosphorylate additional proteins, proteins, and thus,thus, a cascade cascade of protein interactions interactions is is established established FIGURE 1.5 FIGURE 1.5Drawing Drawing illustrating illustrating an epithelial— an epithelial- that ultimately activatesaatranscription ultimately activates transcription factor. mesencnymal interaction. mesenchymal interaction. Following Following anan initial sig— initial sig- This transcription factor then activates This activates oror inhib- inhib- nal from one nal one tissue, a second second tissue tissue is is induced induced to its gene expression. expression. TheThe pathways pathways areare numerous numerous differentiate into a speci differentiate specificc structure. structure. The The first tis— rst tis- sue sue constitutes inducer, and constitutes the inducer, and the second second is is the and complex and in some cases are are characterized characterized responder. Once responder. Once the the induction process is induction process is initiated, initiated, by one one protein protein inhibiting another that in turn turn ac- ac- signals signals [arrows] (arrows) are are transmitted transmitted inin both directions directions tivates another protein (much like the situation tivates situation differentiation process. to complete the differentiation process. with hedgehog signaling). fi fi fi fi  Chapter1- Chapter Introduction to 1 Introduction to Molecular Molecular Regulation Regulation and Signaling Signaling 7 ——-- Ligand Ligand Ligand — Receptor Receptor complex Cell membrane Cell membrane P Activated Activated (kinase) region (kinase) region Nuclear P - Activated protein Cytoplasm pores P Activated proteir Activated protein complex Activated Activated protein Activated protein P -complex complexacts acts as a as a transcription factor transcription factor Nucleus Nucleus FIGURE FIGURE1.6 1.6 Drawing Drawingof ofaatypical typical signal signaltransduction transduction pathway pathway involving involving aa ligand ligand and receptor. Activation and its receptor. of the receptor is is conferred conferred by by binding binding to to the ligand. ligand. Typically, Typically, the the activation activation is enzymatic involving a tyrosine kinase, kinase, although although other enzymes may other enzymes may be be employed. employed. Ultimately, Ultimately, kinase kinase activity results results in aa phosphorylation phosphorylation cascade of cascade of several several proteins proteins that that activates aa transcription factor factor for for regulating