euk DNA packaged w proteins to form

chromatin

nucleosome

nucleosomes contain DNA wrapped around protein core of 8 histone mlcs

Reg gene expression in Euk

30 nm chromatin fiber

transcriptional machinery deal w DNA packing: Chromatin remodeling, histone mod

Histone mod: Histone N-

term tails can interact w: DNA, other

histones, other proteins in

nucleus

Reg gene expression: mlclr basis determination Reg Euk transcription more complicated by packing DNA into chromatin, complex DNA w proteins. basic unit chromatin nucleosome, strand of DNA wrapped 1.7 x (147 nucleotide base pairs) around octomeric histone core 2 copies each 4 distinct histone proteins. basic struct can be packaged into more complex struc, ie 30 nm fibers in interphase nucleus & highly packed metaphase chromosome. State packing chromatin will affect accessibility transcriptional machinery to DNA. Euk evo mech to mod condensation state chromatin to reg gene expression. include ATP-dependent multi-protein machines chromatin remodeling complexes, histone mods (methylation, acetylation phosph). Mod funct as complex codes to direct proteins to particular regions DNA. depend code, (de)condense (pack) chromatin in region. Transcriptional activators interact directly w transcriptional machinery but recruit histone-mod enz or chromatin-remodeling complexes to genes they reg to facilitate transcriptional (in)activation. Cells remember history thru inherited patterns of gene expression (mlclr basis determination) Inherited patterns gene expression: epigenetic inheritance contribute: chromatin state via heritable histone mods, DNA methylation, pos feedback loops.

Histone mod: histone tails

MM methylationMA acetylation MP phosphorylation

Histone mod enz: add/ remove each mod; all reversible

newly exposed DNA

chromatin remodeling

How histone mod ∆ chromatin struct? prevent assoc taiils w DNA & core histones, making DNA more accessible to transcription factors or remodeling enz recruit other proteins: chromatin remodeling complexes & general transcription factors (TFIID) closed vs. open chromatin

transcriptional activators can direct assembly

transcription complex

Histone mod enzlong-lasting ∆ in gene expression thru inherited histone mod

transcriptional activators can direct local alterations in

chromatin struc Mlclr basis determination: how cells remember devel decisions

Epigenetic inheritance: heritable diff phenotype cell not from ∆ nuc seq DNA. chromatin state (via heritable histone mod), DNA methylation, pos feedback loops

DNA methylation turns genes off by attracting proteins that block gene expression

long-lasting ∆ in gene expression thru inherited DNA methylation

long-lasting ∆ in gene expression thru pos feedback

loops

Protein A: transcriptional regulator activates own

transcription

Pattern formationin Drosophila

Drosophila melanogaster: Easy to culture, grows in bottles w yeast for food rapid devel 24 hrs to hatch Simple genetics 4 chromosomes 14,000 genes sophisticated genetic and molecular biology tools > century genetics combined w tools for isolating DNA corresponding to genes identified by mutation. Completely sequenced genome.

segments of Drosophila larva

syncitium multiple nuclei in common cytoplasm blastoderm insect blastula

syncytial blastoderm

cellular blastoderm

transcriptional regulators intercellular signaling

communication between nuclei

contents egg: maternal pronucleus, DNA (meiosis), nutrients (yolk), transcripts (mRNA), proteins

Maternal to zygotic transition

Sperm

activation of maternal proteins & mRNAs

fertilization

egg activation

cell division

early asymm(only some orgs)

beginning of zygotic transcription

new zygotic proteins made degradation maternal prod

EMBRYO IS ON ITS OWN

genetic control early embryonic devel: Nobel physiology/ med 95. Christiane Nusslein-Volhard, Eric Wieschaus: results published 1980

maternal-to-zygotic transition in diff orgs

↯mutagen

select for mutations that cause lethality & abnormal patternMutagenesis screen

follicle cells generate asymm

mRNAPROTEIN

Nanos and Bicoid mRNAs produce protein gradients

egg polarity genes

organization egg polarity gradient systems Bicoid transcription regulator Nanos translation regulator Torso

receptor tyrosine kinaseBicoid acts as graded morphogen

PHENOTYPES MUTANTS

GAP GENES

EXPRESSIONGENE/S

PAIR RULE GENESSEGMENT POLARITY GENES

mutations that affect

anterior/posterior patterning

Most these genes encode transcription regulators

EGG POLARITY protein bicoid binds GAP gene enhancers

GAP bindPAIR RULE

PAIR RULE bindSEGMENT POLARITY

GAP GENE EXPRESSION

PAIR RULE GENE EXPRESSION

if mutated

if mutated

if mutated

normal embryo (or wild type)

embryo mutant for Hunchback

embryo mutant for Giant

embryo mutant for Kruppel

pair rule gene expression in gap mutants

Broad overlapping

bands

7 stripes

14 stripes

Anterior gradient

gene regulatory cascade specifies segments

body of larvae made up 14 segments, each can ID by particular pattern of hairs. early devel of Drosophila (& other insects) differs most vert: embryo doesn’t cleave into individ cells. period during which nuclei proliferate in common cytoplasm. multiple nuclei share common cytoplasm syncytium. certain # divisions, most of nuclei move to periphery of cell, form single layer nuclei, each barely separated from neighbors by a slight invagination of the plasma membrane. stage of development syncytial blastoderm. After a few additional nuclear divisions, each nucleus becomes completely enclosed by membranes in cellularization. stage of devel after cellularization and before gastrulation is called the cellular blastoderm stage. Animal development relies on: genome of mother, which directs the production of the oocyte, and the embryonic genome that results from fusion of haploid sperm & egg nucleus. Depending on animal, maternal genome can direct more or less of early devel. flies, maternal component significant mammals maternal component is very minor. Genetic analysis combined w newly developed molecular biology tools made it possible to learn molecular mechanisms that underlie Drosophila devel. 3 groups maternally-expressed genes alter anterior/posterior pattern: anterior, posterior, & terminal group. For each mutant class, key gene. Torso, encodes receptor tyrosine kinase activated only at poles of egg during oogenesis by signals sent from subset of follicle cells that surround egg. After activated: cytoplasmic determinant. 2 other key genes encode cytoplasmic determinants: Bicoid anterior Nanos for posterior group. mRNAs for 2 genes loaded into egg in polarized way: Bicoid at anterior, Nanos at posterior tip. After egg fertilized, mRNA translated but stays in place. protein forms gradient, highest at site where mRNA localized. gradient persists thru most of syncytial blastoderm stage. Bicoid encodes transcriptional activator functions as cytoplasmic determinant & morphogen gradient. Evidence functions graded fashion first & from observation protein forms gradient from anterior to posterior & 2 nd, more important, when shape of gradient D by ^/ dec # copies bicoid gene, D spatial pattern embryo in coord manner. 3 groups embryo-expressed genes alter anterior/posterior pattern: gap genes, pair rule genes segment polarity genes. Mutations in gap genes caused large gaps in pattern. other classes mutants surprising bc instead of alterations in contiguous regions embryo, mutations affected alternating regions. pair rule mutants, of 14 segments, 7 missing, alternated w ones remained. Some mutations skipped odd-# segments, others skipped even-# (one called even-skipped). segment polarity mutants also showed an alternating pattern, but differed from pair rule genes: half-segments missing/ duplicated in reverse polarity. Each gene unique pattern expression mirrored mutant phenotypes. Gap genes expressed in broad bands w each gene unique pattern partially overlapped w that of other gap genes; pair rule genes expressed in 7 stripes, some overlapped w other pair rule genes; segment polarity genes expressed in pattern 14 stripes. 9 gap genes respond to gradients of bicoid & nanos & consequences of torso signaling by becoming activated in broad & overlapping regions of syncytial blastoderm stage embryo. gap genes direct the expression of pair-rule genes, & pair-rule genes, in turn, direct expression of segment polarity genes. transcriptional regulatory cascade: very efficient way pattern embryo during syncytial blastoderm stage bc nuclei exposed to common cytoplasm allowing transcription factors to move by diffusion & enter nuclei near nucleus that transcribed them