Seminario 4 Regulación de la expresión génica I - Sebastian Giusti

Introduction to Gene Expression Regulation

Overview of the Course

• Sebastián Justi introduces himself as one of the instructors for virtual classes on molecular biology and genetics, emphasizing the aim to support students through theoretical topics and bibliographic readings.

• He mentions that the course offers optional in-person discussion classes and consultation sessions, wishing students a good experience with the subject.

Examples of Gene Expression Regulation

• The first example discussed is during development, where a single fertilized cell (zygote) undergoes mitotic division into billions of cells, forming an adult organism through cellular interactions and signaling processes.

• It is highlighted that despite sharing the same genome, cells differentiate morphologically, biochemically, and functionally—illustrated by comparing neurons and hepatocytes.

Differential Gene Expression

Cellular Specialization

• Different techniques in molecular biology have shown that neurons and hepatocytes express different messenger RNAs despite having identical genomes.

• Neurons express specific genes related to their function (e.g., neurofilament genes), while hepatocytes express genes like albumin and alcohol dehydrogenase crucial for liver functions.

Housekeeping Genes

• Both cell types also share a subset of housekeeping or constitutive genes necessary for basic cellular functions such as cytoskeletal proteins or chromatin compaction regulators.

Mechanisms Behind Differential Gene Expression

Understanding Mechanisms

• The lecture poses questions about mechanisms explaining why certain genes are expressed in one cell type but not another. This inquiry will be explored throughout the class.

Acute Response to Stimuli

Hormonal Influence on Gene Expression

• A second physiological example involves acute responses to stimuli; specifically, how secretory cells in mammary glands respond to prolactin during late pregnancy by expressing previously unexpressed genes like beta-casein and alpha-lactalbumin.

Conceptualizing Gene Expression

Definition of Gene Expression

• To understand gene regulation better, it’s essential to define gene expression: approximately 30,000 human nuclear genes encode various functional products primarily proteins.

Process of Gene Expression

• A gene is considered expressed when transcription occurs followed by translation. Some functional products are RNA itself (e.g., tRNA or rRNA), which only require transcription for expression.

Mechanisms of Gene Regulation

Overview of Transcription Control

• The first checkpoint in gene regulation is the control that allows for the production of a primary transcript from DNA sequences, influenced by cellular and molecular mechanisms.

• These regulatory mechanisms can be categorized into two classes: pre-transcriptional regulation and transcription itself, which will connect to subsequent processes affecting gene expression and stability.

Chromatin Accessibility

• Understanding how DNA interacts with transcription machinery involves recognizing that RNA polymerase II must access the DNA template, which is largely determined by chromatin compaction levels.

• Chromatin exists in varying degrees of compaction; during mitosis, it reaches maximum condensation. However, not all genomic sites have the same level of accessibility.

• Open or "lax" chromatin states allow transcriptional machinery access, while closed chromatin regions prevent transcription.

Nucleosome Structure and Function

• To grasp the molecular dynamics regulating chromatin condensation and gene expression, one must analyze DNA-histone interactions since nucleosomes are the structural units of chromatin.

• Each nucleosome consists of an octamer formed by histone proteins (H2A, H2B, H3, H4), around which DNA wraps.

Histone Modifications Impacting Gene Expression

• Histones are rich in positively charged amino acids; this positive charge facilitates electrostatic attraction to negatively charged DNA.

• The net positive charge on histones can be modulated within cells to influence their interaction with DNA.

Chemical Modifications of Histones

• Amino-terminal ends of histones protrude from nucleosomes and are subject to various covalent modifications that affect nucleosome interactions and overall chromatin structure.

• Key modifications include acetylation (addition of an acetyl group), methylation (addition of a methyl group), and phosphorylation (addition of a phosphate group).

Functional Implications of Acetylation and Methylation

• Acetylation typically occurs on lysine residues (indicated as 'K') and results in loss of positive charge at physiological pH, reducing electrostatic attraction between histones and DNA.

Understanding Chromatin Compaction Mechanisms

Histone Modifications and Their Effects

• The reduction of the electrostatic attraction between histones and DNA occurs due to modifications like methylation, which does not alter the positive charge of histones.

• Methylated lysines cannot undergo acetylation, blocking their positive charge and often leading to a closed chromatin state with high affinity for DNA.

• Acetylation removes a positive charge from histones, decreasing their electrostatic affinity for DNA; methylation and acetylation are mutually exclusive reactions.

• Enzymes such as histone acetyltransferases mediate lysine acetylation, while deacetylases remove these modifications; similar processes exist for methylation through methyltransferases and demethylases.

• The dynamic interplay of these modifications allows different chromatin regions to exhibit varying levels of compaction at different cellular life stages.

Chromatin Remodeling Complexes

• Chromatin remodeling complexes consist of protein subunits that utilize ATP hydrolysis energy to modify nucleosome positioning or structure.

• These complexes can shift nucleosomes within the genome, making certain genomic segments accessible for transcription machinery.

• They can also remove histone subunits from specific genomic segments, reducing compaction and enhancing immediate accessibility.

• Additionally, chromatin remodelers may exchange standard histones with specialized variants that have differential affinities for DNA.

DNA Methylation's Role in Gene Regulation

• DNA methylation involves adding a methyl group directly onto cytosine residues adjacent to guanine nucleotides (CpG sites), particularly concentrated in promoter regions near gene start points.

• Methylated cytosines inhibit RNA polymerase II binding at promoters, effectively silencing genes by preventing transcription initiation.

• This process also recruits enzymes that modify nearby histones towards a more compact state, further contributing to gene silencing mechanisms.

• DNA methylation patterns can be inherited during mitosis via maintenance methyltransferases that copy these patterns onto newly synthesized strands post-replication.

Epigenetic Regulation Overview

• The combined effects of covalent histone modifications, chromatin remodeling activities, and DNA methylation regulate chromatin compaction levels and transcriptional accessibility across the genome.

Understanding Molecular Biology in Developmental Context

The Role of Molecular Biology in Embryology

• The context of molecular biology is crucial, especially in developmental biology and embryology, where it encompasses broader meanings and environmental regulations influencing genetic factors.

Mechanisms of Chromatin Compaction

• Lax chromatin, which is transcriptionally active, features acetylated and non-methylated histones due to the action of chromatin remodeling complexes that enhance accessibility by displacing or removing nucleosomes.

• In contrast, closed chromatin has deacetylated histones and methylated cytosines. Methylation prevents acetylation, leading to a compacted state that blocks transcriptional access.

Differential Gene Expression

• Understanding differential gene expression between cells with identical genomes can be illustrated through neurons and hepatocytes having distinct chromatin compaction states affecting their gene expression profiles.

• Regions of compacted versus lax chromatin differ between neuron and hepatocyte genomes, allowing selective access to transcription machinery for specific genes.

Epigenetic Mechanisms Influencing Chromatin States

• Local compaction and relaxation of chromatin are mediated by epigenetic mechanisms such as covalent modifications of histones and DNA methylation.

• A key question arises regarding what mechanisms recruit specific genomic regions to epigenetic modifiers that influence local chromatin states.

Regulation of Transcription Initiation

• Even if a region's chromatin is relaxed, this does not guarantee transcription will occur; additional regulatory mechanisms are necessary for effective transcription initiation.

• The central enzyme for transcription is RNA polymerase II (Pol II), which synthesizes RNA from a DNA template but requires activation through regulatory processes.

Characteristics of RNA Polymerase II

• Pol II consists of multiple protein subunits; one critical component is the carboxy-terminal domain (CTD), which plays an essential regulatory role during transcription initiation.

• Pol II remains inactive at the promoter until extensive phosphorylation occurs on serine residues within the CTD, triggering its activation for transcription progression.

Recruitment Mechanisms for Transcription Factors

• Within cells, there are numerous Pol II complexes actively transcribing different genes simultaneously rather than a single complex operating alone.

• For Pol II to bind effectively to a promoter region at the start site of transcription, additional proteins known as basal transcription factors must first recognize and bind to the promoter sequence.

Understanding RNA Polymerase II Activation

The Role of Promoters in Transcription Initiation

• RNA polymerase II is positioned at a promoter region, which is crucial for the initiation of transcription.

• Promoters contain specific sequences, such as the TATA box, characterized by high thymine and adenine content, essential for transcription regulation.

Regulatory Sequences and Their Functions

• Regulatory DNA segments can be located thousands of base pairs away from the transcription start site but still influence gene expression.

• These regulatory regions are classified based on their effects: enhancers increase transcription frequency while silencers decrease it.

Interaction with Transcription Factors

• Specific transcription factors bind to these regulatory sequences to modulate RNA polymerase activity; they differ from basal transcription factors that are conserved across all genes.

• The specificity of these factors allows for tailored gene expression responses depending on cellular conditions.

Mechanisms of Long-Distance Regulation

• Enhancers and silencers can exert effects over long distances due to the ability of DNA to fold, bringing distant elements into proximity during transcription initiation.

• Coactivators interact with specific transcription factors to form a complex that facilitates communication between regulatory elements and RNA polymerase.

Complex Formation and Signal Transduction

• The mediator complex plays a critical role in transducing signals from specific transcription factors to RNA polymerase II, enabling effective phosphorylation necessary for initiating transcription.

• This phosphorylation event is contingent upon the presence of appropriate enhancer signals that activate the carboxy-terminal domain (CTD).

Additional Functions of Specific Transcription Factors

• Beyond forming initiation complexes, specific transcription factors can recruit histone-modifying enzymes or chromatin remodelers to enhance accessibility at promoters.

• Such recruitment may lead to local chromatin decondensation, facilitating access for basal transcription machinery.

Epigenetic Modifications and Gene Expression Control

• Specific transcription factors help position epigenetic modifiers within the genome, influencing which genes are expressed based on chromatin state alterations.

• While present in cells, these factors may remain inactive until external signals trigger their activation through intracellular signaling pathways.

Conclusion: The Dynamic Nature of Gene Regulation

Prolactin and Gene Expression Changes

Prolactin's Role in Gene Expression

• Prolactin induces changes in gene expression within mammary gland secretory cells, suggesting it triggers a signaling cascade.

• This cascade may lead to the phosphorylation of a specific transcription factor, which can then migrate to the nucleus.

• The phosphorylated transcription factor is essential for forming part of the transcription initiation complex that activates specific genes.

Epigenetic Modifications and Transcription Regulation

• Key concepts discussed include how epigenetic modifications regulate chromatin compaction, affecting transcriptional machinery accessibility.

• These modifications are covalent alterations of histones that play a crucial role in gene regulation.

Chromatin Remodeling and DNA Methylation

• The activity of chromatin remodeling complexes and DNA methylation were highlighted as significant factors influencing gene expression.

• Specific transcription factors can recruit epigenetic modifiers to particular genomic sites, facilitating targeted modifications.

Cellular Variability in Gene Expression

• Different cell types exhibit varying repertoires of specific transcription factors, leading to distinct subregions of the genome being either condensed or relaxed.