Regulación de la expresión genética en eucariontes

Regulación de la expresión genética a nivel TRANSCRIPCIONAL

Bibliografía: Alberts MBOTC Cap. 4 y 7

Activadores

Represores

Coactivadores

Factores de transcripción

basal

Promotor Basal: secuencia de nucleótidos necesaria para la fijación de la RNA polimerasa.

Secuencias reguladoras: A)  Intensificadoras (enhancers): secuencias que estimulan la transcripción y cuya

localización puede ser a miles de nucleótidos de distancia "río arriba o abajo" del promotor

B)  Silenciadoras (silencers): secuencias que inhiben la transcripción. También pueden hallarse muy distantes del promotor.

Factores basales de transcripción: complejo proteico que interacciona con el sitio

promotor. Son esenciales para la transcripción pero no pueden aumentar o disminuir su ritmo.

Factores específicos de la transcripción: complejo de proteínas reguladoras que pueden ser activadoras o represoras.

A)  Proteínas activadoras: interaccionan con las secuencias intensificadoras del gen

(enhancers).

B)  Proteínas represoras: interaccionan con las secuencias silenciadoras del gen (silencers).

Los factores de transcripción son proteínas de unión a DNA

Dominio de unión a DNA Dominio transactivador Domino regulable (p.ej. por hormona)

Interacción de los dominios de unión a DNA con sus secuencias específicas

a)  Hélice-vuelta-Hélice

b)  Dedos de Zinc

c)  Cierre de Leucina

d)  Hélice-asa-Hélice

Receptores nucleares a hormonas (factores de transcripción específicos) y sus elementos de

respuesta en el DNA (Enhancer/Silencer)

Receptores a estrógenos, progesterona, testosterona Receptores a glucocorticoides (cortisona, hidrocortisona, dexametasona) Receptores a ácido retinoico, tiroxina y Vitamina D

Promotor Basal

Sin Promotor Basal

Con enhancer Sin Promotor Basal

Con enhancer y Promotor Basal

Con enhancer y Promotor Basal

Con enhancer y Promotor Basal

Con enhancer y Promotor Basal

Relevancia del Enhancer y su Posición respecto al Promotor Basal

32310.7 Chromatin’s role in eukaryotic gene regulation

Figure 10-29 The structure of chromatin. (a) The nucleosomein decondensed and condensed chromatin. (b) Chromatinstructure varies along the length of a chromosome. The leastcondensed chromatin is shown in yellow, regions of intermediatecondensation in orange and blue, and heterochromatin coatedwith special proteins (purple) in red. [Part b from Peter J. Hornand Craig L. Peterson, “Chromatin Higher Order Folding: Wrapping UpTranscription,” Science 297, September 13, 2002, p. 1827,Figure 3. Copyright 2002, AAAS.]

Nucleosomes:the basic unit of chromatin

Chromatin fiber of packednucleosomes

11 nm

30 nm

(a)

Short region ofDNA double helix 2 nm

(b)

reason for the 100- to 1000-fold difference in recombi-nation frequencies in euchromatin compared with het-erochromatin. Euchromatin, with its more open confor-mation, was hypothesized to be more accessible toproteins needed for DNA recombination. Note that, inthese cases, chromatin has a passive role: regions of thegenome are either open or closed, and transcription andrecombination are largely restricted to regions of openchromatin. However, observations of three phenomenabegan to change this view. These phenomena demon-strated that euchromatic chromatin could be alteredand, more importantly, that active genes in these regions

could become inactive. The three phenomena are X in-activation, imprinting, and position-effect variegation.

MESSAGE The chromatin of eukaryotes is not uniform.Highly condensed heterchromatic regions have fewer genesand lower recombination frequencies than do the lesscondensed euchromatic regions.

X inactivation in female mammalsIn Chapter 15, you will learn about the effects of genecopy number on the phenotype of an organism. Fornow, it is sufficient to know that the number of tran-scripts produced by a gene is usually proportional to thenumber of copies of that gene in a cell. Mammals, forexample, are diploid and have two copies of each genelocated on their autosomes. However, as discussed inChapter 2, the number of the X and Y sex chromo-somes differs between the sexes, with female mammalshaving two X chromosomes to only one in males. Themammalian X chromosome is thought to contain about1000 genes. Females have twice as many copies of theseX-linked genes and would normally express twice asmuch transcript from these genes as males. (Not havinga Y chromosome is not a problem for females, becausethe very few genes on this chromosome are only re-quired for the development of males.) This dosage im-balance is corrected by a process called dosage compen-sation, which makes the amount of most gene productsfrom the two copies of the X chromosome in femalesequivalent to the single dose of the X chromosome inmales. This equivalency is accomplished by random inac-tivation of one of the two X chromosomes in each cellat an early stage in development. This inactive state isthen propagated to all progeny cells. (In the germ line,the second X becomes reactivated in oogenesis). The in-activated chromosome, called a Barr body, can be seenin the nucleus as a darkly staining, highly condensed,heterochromatic structure.

Two aspects of X-chromosome inactivation are rele-vant to a discussion of chromatin and the regulation ofgene expression. First, most of the genes on the inacti-vated X chromosome are turned off (they are said to besilenced ). In organisms where this phenomenon is bettercharacterized, one can see that alteration in the chro-matin structure of this chromosome has silenced previ-ously active genes. Second, genes on the inactivated chro-mosome remain inactive in all descendants of these cells.Such a heritable alteration, in which the DNA sequenceitself is unchanged, is called epigenetic inheritance.

A beautiful example of X inactivation is the patternof fur color on the calico cat. Because the same X chro-mosome is turned off in all descendants of a cell, large

44200_10_p301-340 3/9/04 1:05 PM Page 323

Los diferentes estados de la CROMATINA

La expresión genética en eucariontes requiere de cambios en el estado de la cromatina

Desacetilasas de Histonas

(HDACs)

Acetilasas de Histonas (HATs)

Complejos remodeladores de cromatina

THE REGULATION OF CHROMATIN STRUCTURE 223

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119

N-terminal tails

phosphorylation

top vlew

a acetylationO methylat ionS phosphorylat ion

ubiq ui ty lat ionI acetylation or methylation

Figure 4-4O A map of histone modificationson the surface of the nucleosome coreparticle. As noted, the histone tails havebeen omitted here (compare with Figure4-39). The functions of most of these coremodifications are not yet known. (Adaptedfrom M.S. Cosgrove, J.D. Boeke andC. Wolberger, Nat. Sttuct. Mol. Biol.1 1 :1037-1043, 2004. With permission fromMacmil lan Publishers Ltd.)

P ffi acetvlation UI

Sl"brbr.domains

ubiquitylation

(A)

M methylationI

(B)

Figure 4-39 The covalent modif icat ion of core histone tai ls. (A) The structure of the nucleosome highl ighting the locationof the f irst 30 amino acids in each of i ts eight N-terminal histone tai ls (green). (B\ Well-documented modif icat ions of thefour histone core proteins are indicated. Although only a single symbol is used for methylat ion here (M), each lysine (K) orarginine (R) can be methylated in several dif ferent ways. Note also that some posit ions (e.9., lysine 9 of H3) can be modif iedeither by methylat ion or by acetylat ion, but not both. Most of the modif icat ions shown add a relat ively small moleculeonto the histone tai ls; the exception is ubiquit in, a 76 amino acid protein also used for other cel l processes (see Figure6-92). (Adapted from H. Santos-Rosa and C. Caldas, Eur. J. Cancer 41:2381-2402,2005. With permission from Elsevier')

The modifications of the histones are carefully controlled, and they haveimportant consequences. The acetylation of lysines on the N-terminal tailstends to loosen chromatin structure, in part because adding an acetyl group tolysine removes its positive charge, thereby reducing the affinity of the tails for

H3 t a i l s

- t

I

bottom view

side v iew

El código de histonas (Modificaciones postraduccionales)

226 Chapter 4: DNA, Chromosomes, and Genomes

(A)

M M

R K2 4

MMl P

AM

YI?A A AM v r l MI r l

K R K K14 1118 23

K S9 1 0

R K S K262728 36

Mr

K79

Figure 4-44 Some specific meanings ofthe histone code. (A) The modif icat ionson the histone H3 N-terminal tai l areshown, repeated from Figure 4-39.(B) The H3 tai l can be marked by dif ferentcombinations of modif icat ions thatconvey a specif ic meaning to the stretchof chromatin where this combinationoccurs. Only a few of the meanings areknown, including the four examplesshown. To focus on just one example, thetr imethylat ion of lysine 9 attracts theheterochromatin-specif ic protein HP1,which induces a spreading wave offurther lysine 9 tr imethylat ion fol lowedby further HP1 binding, according to thegeneral scheme that wi l l be i l lustratedshortly (see Figure 4-46). Not shown isthe fact that, as just implied (see Figure4-43), reading the histone code general lyinvolves the joint recognit ion of marks atother sites on the nucleosome along withthe indicated H3 tai l recognit ion. Inaddit ion, specif ic levels of methylat ion(mono-, di-, or tr i-methyl groups) arerequired, as in Figure 4-42.

(B ) modification state "meaning"

heterochromat in format ion,gene s i l enc i ng

gene expreSsron

gene expreSSton

si lencing of Hox genes,X chromosome inact ivat ion

The marks on nucleosomes due to covalent additions to histones aredynamic, being constantly removed and added at rates that depend on theirchromosomal locations. Because the histone tails extend outward from thenucleosome core and are likely to be accessible even when chromatin is con-densed, they would seem to provide an especially suitable format for creatingmarks in a form that can be readily altered as a cell's needs change. Althoughmuch remains to be learned about the meaning of the many different histonecode combinations, a few well-studied examples of the information that can beencoded in the histone H3 tail are listed in Figure 4-44.

A Complex of Code-reader and Code-writer proteins Can SpreadSpecific Chromatin Modifications for Long Distances Along aChromosomeThe phenomenon of position effect variegation described previously requiresthat at least some modified forms of chromatin have the ability to ipreud fotsubstantial distances along a chromosomal DNA molecule (see Figure 4-36).How is this possible?

The enzymes that modify (or remove modifications from) the histones innucleosomes are part of multisubunit complexes. They can initially be broughtto a particular region of chromatin by one of the sequence-specific DNA-bind-ing proteins (gene regulatory proteins) discussed in chapters 6 and 7 (for a spe-cific example, see Figure 7-87). But after a modifying enzyme "writes" its markon one or a few neighboring nucleosomes, events that resemble a chain reactioncan ensue. In this case, the "code-writer" enzyme works in concert with a code-reader protein located in the same protein complex. This second protein con-tains a code-reader module that recognizes the mark and binds tightly to thenewly modified nucleosome (see Figure 4-42), positioning its attached writerenzyme near an adjacent nucleosome. Through many such read-write cycles,the reader protein can carry the writer enzyrne along the DNA-spreading themark in a hand-over-hand manner along the chromosome (Figure 4-45).

In reality, the process is more complicated than the scheme just described.Both readers and writers are part of a protein complex that is likely to contain

M

N

9

M Ar l

K K4 9

P At l

S K1 0 1 4

MI

K27

¿Cómo se interpreta el código de Histonas?

THE REGULATION OF CHROMATIN STRUCTURE

Many of the combinations appear to have a specific meaning for the cellbecause they determine how and when the DNA packaged in the nucleosomesis accessed, leading to the histone code hlpothesis. For example, one type ofmarking signals that a stretch of chromatin has been newly replicated, anothersignals that the DNA in that chromatin has been damaged and needs repair,while many others signal when and how gene expression should take place.Small protein modules bind to specific marks, recognizing for example a tri-methylated lysine 4 on histone H3 (Figure tl-42). These modules are thought toact in concert with other modules as part of a code-reader complex, so as toallow particular combinations of markings on chromatin to attract additionalprotein complexes that execute an appropriate biological function at the righttime (Figure 443).

cova len tmodi f icat ionon histone ta i l(mark)

CODE READERBINDS ANDATTRACTS OTHERCOMPONENTS protein complex wi th

catalytic activities andaddi t ional b inding s i tes

225

Zn

Figure 4-42 How each mark on anucleosome is read. The structure of aprotein module that sPecif ical lYrecognizes histone H3 tr imethylated onlysine 4 is shown. (A) Space-f i l l ing modelof an ING PHD domain bound to ahistone tail (green, with the trimethylgroup highl ightedin yel low). (B) A r ibbonmodel showing how the N-terminal sixamino acids in the H3 tai l are recognized.The doshed Iines represent hydrogenbonds. This is one of many PHD domainsthat recognize methylated lysines onhistones; dif ferent domains bind t ightlyto lysines located at different positions,and they can discriminate between amono-, di-, and tr i-methylated lysine. In asimilar way, other small protein modulesrecognize specif ic histone side chainsthat have been marked with acetylgroups, phosphate grouPs, and so on.(Adapted from P.V. Pena et al., Nature442:100-1 03, 2006. With permission fromMacmil lan Publishers Ltd.)

Figure 4-43 Schematic diagram showinghow the histone code could be read by acode-reader complex. A large proteincomplex that contains a series of proteinmodules, each of which recognizes aspecif ic histone mark, is schematical lyillustrated (green).This "code-readercomplex" wil l bind t ightly only to a regionof chromatin that contains several of thedifferent histone marks that it recognizes'Therefore, only a specific combination ofmarks wil l cause the complex to bind tochromatin and attract addit ional proteincomplexes (purple)that catalyze abiolooical function.

(B)

orotein modules scaffoldbinding to speci f ic proteinhistone modi f icat ionson nucteosome

Lectura del código de histonas Transmisión de la información

Remodelación de cromatina Activación/Silenciamiento Transcripcional

Secuencia de eventos en la activación transcripcional

HOW GENETIC SWITCHES WORK

(A) rN SOLUTTON

to)

(B) ON DNA

coactivator\ ACTIVATES

PTION

.****liaGENE ON

coactivator ACTIVATESTRANSCRIPTION

II

447

Figure 7-51 Eucaryotic Aene regulatoryproteins often assemble into complexeson DNA. Seven gene regulatory proteinsare shown in (A). The nature and functionof the complex they form depends on thespecif ic DNA sequence that seeds theirassembly. In (B), some assembledcomplexes activate gene transcript ion,while another represses transcript ion.Note that both the red andthe greenproteins are shared by both activating andrepressing complexes. Proteins that do notthemselves bind DNA but assemble onother DNA-bound gene regulatoryoroteins are often termed coactivators orco-reoressors. However, these terms aresomewhat confusing because theyencompass an enormous variety ofproteins including histone readers andwriters, chromatin remodeling complexes,and many other classes of proteins. Somehave no intr insic act ivi ty themselves butsimply serve as a "scaffolding"to attractthose that do.

activates+ . transcfl pI|on

Figure 7-52 Schematic depict ion of acommittee of gene regulatory proteinsbound to an enhancer. The proteinshown in yel lowis cal led an architecturalprotein since i ts main role is to bend theDNA to al low the cooperative assemblyof the other components. The structuredeoicted here is based on that found inthe control region of the gene that codesfor a subunit of the T cel l recePtor(discussed in Chapter 25), and i t act ivatestranscription at a nearby promoter. Onlycertain cel ls of the developing immunesystem, which eventually give rise tomature T cells, have the complete set ofDroteins needed to form this structure.

GENE OFF

coactivator\ ACTIVATES

PTION

GENE ON GENE ON

Typically, a few relatively short stretches of nucleotide sequence guide theassembly of a group of regulatory proteins on DNA (see Figure 7-51). However,in some extreme cases of regulation by committee a more elaborateprotein-DNA structure is formed (Figure 7-52). Since the final assemblyrequires the presence of many gene regulatory proteins that bind DNA, it pro-vides a simple way to ensure that a gene is expressed onlywhen the cell containsthe correct combination of these proteins. We saw earlier how the formation ofheterodimers in solution provides a mechanism for the combinatorial control ofgene expression. The assembly of complexes of gene regulatory proteins onDNA provides a second important mechanism for combinatorial control, onethat offers far richer opportunities.

Compf ex Genetic Switches That Regulate DrosophiloDevelopment Are Built Up from Smaller ModulesGiven that gene regulatory proteins can be positioned at multiple sites alonglong stretches of DNA, that these proteins can assemble into complexes at eachsite, and that the complexes influence the chromatin structure as well as therecruitment and assembly of the general transcription machinery at the pro-moter, there would seem to be almost limitless possibilities for the elaborationof control devices to regulate eucaryotic gene transcription.

A particularly striking example of a complex, multicomponent geneticswitch is that controlling the transcription of the Drosophila Euen-skipped (Eue)gene, whose expression plays an important part in the development of theDrosophila embryo. If this gene is inactivated by mutation, many parts of theembryo fail to form, and the embryo dies early in development. As discussed inChapter 22, at the stage of development when Eue begins to be expressed, theembryo is a single giant cell containing multiple nuclei in a common cytoplasm.This cytoplasm is not uniform, however: it contains a mixture of gene regulatoryproteins that are distributed unevenly along the length of the embryo, thus pro-viding positional information that distinguishes one part of the embryo fromanother (Figure 7-53). (The way these differences are initially set up is discussedin Chapter 22.) Although the nuclei are initially identical, they rapidly begin toexpress different genes because they are exposed to different gene regulatoryproteins. The nuclei near the anterior end of the developing embryo, for exam-ple, are exposed to a set of gene regulatory proteins that is distinct from the setthat influences nuclei at the posterior end of the embryo.

The regulatory DNA sequences controlling the Eue gene are designed to readthe concentrations of gene regulatory proteins at each position along the lengthof the embryo and to interpret this information in such a way that the Eue geneis expressed in seven stripes, each initially five to six nuclei wide and positionedprecisely along the anterior-posterior axis of the embryo (Figure 7-54). How isthis remarkable feat of information processing carried out? Although not all ofthe molecular details are understood, several general principles have emergedfrom studies of Eue and other Drosophila genes that are similarly regulated.

Señalización para la activación de los factores de transcripción específicos (CASO I)

Receptor a Glucocorticoides (GR)

El ligando que estimula (cortisol) es liposoluble y entra a la célula. El GR se encuentra en complejo inactivo con Hsp y es liberado por el ligando. Entra a núcleo donde se dimeriza y actúa sobre sus elementos de respuesta estimulando la transcripción de genes específicos.

Stat1α

En este caso el ligando NO entra a la célula y su receptor es una proteína de membrana. Al unir el ligando, el receptor se activa por fosforilación y actúa como cinasa fosforilando a Stat1α en el citosol. Esta fosforilación promueve la dimerización y entrada al núcleo del factor transcripcional.

Señalización para la activación de los factores de transcripción específicos (CASO II)

Los activadores pueden actuar de manera conjunta

Formas de acción de la proteína activadora

Competencia con el Represor por el mismo sitio de unión

Enmascaramiento del dominio de transactivación

Formas de acción de la proteína activadora

Interacción directa con factores de transcripción

Formas de acción de la proteína activadora

Complejos formados in situ sobre el DNA

Cada gen tiene una combinación particular de intensificadores y silenciadores. Genes distintos pueden compartir idénticas secuencias intensificadoras y silenciadoras, pero no existen dos genes que posean la misma combinación de estas secuencias reguladoras.

Regulación en el desarrollo por proteínas homeóticas (Embriones de Drosophila)

Activadores transcripcionales Represores transcripcionales

Expresión del gen Eve

La zona de regulación involucra activadores y represores

activadores

represores

En la zona de la banda 2 se dan las condiciones de represor y activador

Regulación de la expresión genética a nivel POST-TRANSCRIPCIONAL

Cambios en la expresión genética a nivel TRADUCCIÓN

A B B B B B B B B

B B B B B B B B

¡ Regulación traducción !

Durante el desarrollo embrionario de Drosophila ocurre la traducción selectiva de mRNAs en el embrión para determinar la polaridad del cuerpo de la mosca

Traducción activa

Traducción inactiva

Traducción inactiva Traducción

inactiva

Regulación por grupo HEMO en eritrocitos

En ausencia de HEMO se activa una proteína cinasa que fosforila a eIF2. eIF2 fosforilado no es capaz de intercambiar el GDP por GTP, ya que queda formando complejo inactivo con el eIF2B. Hay depleción de complejo ternario e inhibición de la traducción.

Regulación por siRNAs Presencia de RNA de doble cadena (por virus, RNA anti-sentido, etc.)

Se generan siRNAs de 21 nt por corte con DICER

Se forma RISC cargado con siRNA y varias proteínas incluyendo ARGONAUTA

RISC dirige el siRNA a su mRNA blanco con el cual forma complementariedad PERFECTA y promueve el corte y degradación de este mRNA

Regulación por miRNAs Los miRNAs se transcriben por la RNA polimerasa II como transcritos primarios y son procesados por DROSHA en el núcleo Luego son exportados al citoplasma y son cortados por DICER a tamaño de 21 nt Los miRNAs son tomados por RISC con ARGONAUTA y son dirigidos a sus mRNA blanco con los cuales muestran complementariedad IMPERFECTA generalmente en sus regiones 3’UTR RISC cargado con miRNAs dirige la inhibición traduccional de los mRNAs blanco

Control transcripcional

A- Factores de transcripción B- Grado de condensación de la cromatina C- Grado de metilación del ADN

Control procesamiento del ARNm

Empalme alternativo Grado de poliadenilación

Control transporte del ARNm

Mecanismos que determinan si el ARNm maduro sale o no a citosol

Control traduccional

Mecanismos que determinan si el ARNm presente en el citosol es o no traducido

Control de la degradación del ARNm

Mecanismos que determinan la supervivencia del ARNm en el citosol

Control de la actividad proteica

Mecanismos que determinan la activación o inactivación de una proteína, así como el tiempo de supervivencia de la misma.

RESUMEN