Epigenetic Regulation in Stem Cells

Epigenetic Regulation in Stem Cells

• Stem cells regulate fate-determining gene programs through epigenetic alterations of chromatin.
• Transcription factors rely on binding to co-factor proteins that change chromatin structure and gene activity through epigenetic modifications.

What is Epigenetic?

• Epigenetics studies changes in gene expression or cellular phenotype caused by mechanisms other than changes in DNA sequence.
• Epigenetics refers to functional modifications to the genome that do not involve changes in the nucleotide sequence.
• Epigenetics is described as the "fifth letter" of the DNA code in the genome.
• Stem cells manage their status through multiple layers of molecular events, imposing flexible but precise control over regulatory gene expression.
• Epigenetic changes can modify the expression of many genes involved in stem cell pluripotency maintenance.

Epigenetic Changes: DNA Methylation, Histone Modification, miRNA Regulation

• Epigenetic changes regulate gene expression without altering the DNA sequence.
• Epigenetic changes include:
  • DNA methylation
  • Histone modification
  • MicroRNA (miRNA) regulation

Transcriptional Factor Network

• Extracellular signals influence transcriptional factor networks, impacting gene activity.
• Active genes have active chromatin and are present in pluripotent and differentiated cells.
• Poised genes have bivalent chromatin and reside in pluripotent cells.
• Silent genes have silent chromatin and are present in pluripotent and differentiated cells.
• Nuclear positioning, DNA methylation, histone modification, and non-coding RNA contribute to the epigenetic signature of the cell.
  • Histone modification includes mechanisms like acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and proline isomerization.

DNA Methylation

• In mammals, 5'-methylcytosine (5MeC) accounts for approximately 1% of total DNA bases.
• 5MeC affects 70–80% of all CpG dinucleotides in the genome.
• There are approximately 29,000 CpG islands in the human genome, with over 60% of human genes associated with them.
• Most CpG islands are unmethylated throughout development and in all tissue types.
• DNA methylation patterns are dynamic and vary during development and across the genome.
• The cycle of early embryonic demethylation followed by de novo methylation is critical for determining cell-specific DNA methylation patterns.
• Methyl groups are added to the cytosine of DNA, mainly at CpG sites on the gene promoter.
• It is important in embryogenesis, development, genomic imprinting, and regulation of gene transcription.
• Unmethylation leads to activation, while methylation leads to inactivation.

DNA Methylation

• In the pre-implantation embryo, almost the entire genome is stripped of DNA methylation.
• The genome-wide methylation pattern is re-established through de novo methylation after implantation.
• This pattern is maintained during all following cell divisions.
• Three Requirements:
  • Methylation marks must be established and maintained.
  • They must be read and translated into functional biological information.
  • Removed if necessary.
• DNA (cytosine-5) methyltransferases (DNMTs) catalyze the covalent addition of a methyl group to an unmethylated cytosine.
• Mammals have five DNMTs (DNMT1, 2, 3a, 3b, and 3l), classified into three families based on structural and functional differences.
• DNMT3a and DNMT3b are major de novo methyltransferases:
  • They establish the initial addition of methyl-groups onto CpGs in unmethylated DNA double strands.
  • Their patterns parallel the re-establishment of post-implantation DNA methylation during embryogenesis.
• DNA methylation is associated with transcriptionally silent chromatin.
• It is thought to inhibit gene transcription by two principles:
  • Methylated CpGs inhibit interaction of transcriptional activators in regulatory DNA elements.
  • Methyl-CpGs are bound by specialized proteins that recognize and recruit silencing cofactors.
• Methyl-CpG binding domain protein (MBD) favors regions of higher CpG density and identifies the greatest proportion of CpG islands.
• DNA methylation can be lost passively during DNA replication—maintenance methylation is inhibited.
• Can be actively enzymatically removed.

Mapping DNA Methylation Marks

• The epigenome is highly dynamic and can undergo remodeling during cellular development and diversification.
• 5mC marks are unevenly distributed at the genome.
• 5mCs appear to have characteristic localizations, remodeled in cancer.
• Distribution of DNA modification provides insight into epigenetic information that controls cell biology during development and disease.
• To understand the biological role of DNA modifications in the genome, 5mC is assessed by bisulphite-treatment of DNA.
• Bisulphite-treatment deaminates unmethylated Cs into Uracil (U), followed by standard molecular biology analyses like PCR and massive parallel sequencing.
• Bisulphite-treatment allows for global profiling of 5mC marks.

Programming of DNA Methylation Patterns

• In early embryogenesis, DNA is largely devoid of methylation.
• Post-implantation, de novo methylation begins, mediated primarily by DNA (cytosine-5-)-methyltransferase-3alpha (DNMT3A) and -3beta (DNMT3B).
• When methylation affects CpG islands, methyl-binding proteins trigger a silencing cascade involving histone H3K9 deacetylation and methylation.
• Methyl-binding proteins allows heterochromatin protein 1 (HP1) to bind, eventually resulting in closed chromatin.
• After DNA replication, newly synthesized DNA is unmethylated.
• DNMT1 rapidly scans DNA and deposits methyl groups on newly synthesized DNA, opposite methyl groups on the old DNA strand.
• This results in replication of methylation patterns and silencing maintenance
• Adult methylation patterns are erased in early embryogenesis by epigenetic reprogramming.

DNA Methylation Reprogramming

• The epigenetic genome-wide reprogramming cycle involves two phases of DNA erasure in mammals.
• First, DNA demethylation occurs in male/female primordial germ cells (PGC) of the F1, including imprinted genes.
• The gamete genome undergoes de novo methylation with maternal methylation marks established later than paternal.
• Second, DNA demethylation occurs after fertilization in the F2 zygote, with quicker demethylation in the paternal than maternal genome.
• Paternal and maternal imprinted genes maintain their methylation pattern through preimplantation reprogramming, allowing inheritance of parent-specific monoallelic expression in somatic tissues of the F2.
• Genome-wide remethylation occurs in parental genomes at implantation.
• The timing of DNA demethylation/remethylation waves differs in male/female genomes.
• Early embryonic development is an epigenomic reprogramming step prone to environmental impacts.
• Early development-environment has greater impact on adult phenotype than later-life experiences.

Histone Modification

• Histone modification acts in diverse biological processes such as gene regulation, DNA repair, and spermatogenesis.
• Eukaryotic DNA is densely packed in chromatin to fit into the nucleus which must be flexible for spatiotemporal access to DNA by regulatory proteins.
• Modification involves control of transcription by altering chromatin structure.
• DNA compaction must be dynamic to allow local switching between "open" and "closed" states.
• Histone modification includes: acetylation, methylation, ubiquitination, sumoylation and phosphorylation.
• The smallest packaging unit is the nucleosome, consisting of an octamer of core histones (two each of H2A, H2B, H3, and H4), around which 147 bp of DNA are wrapped in super-helical turns.
• The nucleosome is a dynamic signaling molecule functioning in gene expression states.
• Gene expression states are influenced by methylation and acetylation at the N-terminal histone tails.
• Addition or removal of acetyl- or methyl-groups is conducted by enzymes: histone acetyltransferases/deacetylases and histone methyltransferases/demethylases.
• Histone H3 modification includes methylation which occurs at lysine (K) or arginine (R) residues.
• Methylation is associated either with active or repressed chromatin, depending on the position of the lysine or arginine that is methylated.
• Lysines can be mono-, di-, or trimethylated.
• Trimethylation of H3K4 is often found at actively transcribed gene promoters, while active enhancers are marked by monomethylation (not trimethylation) of H3K4.
• Histone H3 acetylation is generally linked to transcriptional activation.
• Histone tails are positively charged which is neutralized through acetylation that reduces binding to DNA thus allows access for transcriptional activator proteins.
• Lysine acetylation/deacetylation can alter DNA binding, activation, stability, nuclear localization, and coactivator interaction of non-histone transcriptional activators.
• Acetylation of lysine residues by histone acetyltransferases (HATs) use Coenzyme A (CoA) as a donor to transfer an acetyl group to histones.

Epigenetic Control of Hematopoietic Stem Cells

• The key challenge for HSCs is balancing self-renewal with refilling the demand for new blood cells.
• Epigenetic mechanisms play a central role in stem cell fate decisions during normal hematopoiesis and leukemia.
• HSCs can differentiate into lymphoid (Lym) or myeloid/erythroid (My/Er) cells, or after malignant transformation, form acute myeloid (AML) or lymphoid (ALL) leukemias.
• ES cells contain bivalent chromatin structures, with activating H3K4me3 and repressive H3K27me3 histone marks existing on the same locus.
• Bivalent structure poises genes for differentiation, allowing rapid activation (losing H3K27me3) or permanent silencing (losing H3K4me3).

Bivalent Modifications in ES Cells

• A bivalent gene containing both H3K4me3 and H3K27me3 epigenetic modifications in the same area plays a role related to pluripotency in embryonic stem (ES) cells.
• Methylations are catalyzed by Trithorax- and Polycomb-group proteins and play roles specific to lineage development.
• Trithorax-associated H3K4 trimethylation (H3K4me3) is a regulation for transcription which recruits nucleosome remodeling enzymes/histone acetylases.
• Polycomb-associated H3K27 trimethylation (H3K27me3) negatively regulates transcription promoting a condensed chromatin structure.
• Polycomb-group complex, originally found in Drosophila, represses Hox genes and results in transformation of body segments when mutated.
• PCG transcriptional repressors are organized in two main PRCs, PRC1 and PRC2.
• PRC1 recognizes the trimethylated H3K27 (H3K27me3) mark, bringing nucleosomes into PRC2, facilitating widespread methylation over extended regions.
• Polycomb repressive complex 2 (PRC2) stabilize through function with histone methyltransferases is responsible for tri-met H3K27 mark on the chromatin.
• TrxG proteins are evolutionarily maintained chromatin regulators.
• Proteins work as part of large multiprotein complexes that have modified histone or nucleosome-remodeling activities which promote gene expression.

miRNA Regulation

• miRNA regulation involves small non-coding RNA molecules (~22 nucleotides.)
• miRNA Regulation is well conserved in eukaryotic organisms regulation.
• miRNA Regulation functions in transcriptional and post transcriptional regulation of gene expression by base pairing with complementary sequences which silences genes.
• Interplay between microRNA and transcription factors contribute in maintaining a pluripotent state.
• miR-145 regulates OCT4, SOX2 and Klf4 posttranscriptionally, which is highly expressed in differentiated cells, reduced in ES and iPS cells.
• The miR-302/367 cluster is transcriptionally modulated by OCT4 and higher in undifferentiated cells.
• CMYC up-regulates miR-17/92 family, affecting TGF-β signaling on cellular differentiation.

ESC Characterization

• Testing is done to determine whether human embryonic stem cells are pluripotent
• Manipulating the cells to differentiate to form cells characteristic of the three germ layers.
• Injecting them the into a mouse with a suppressed immune system (Teratoma formation) in order to have benign cells.

Research application of ESC

• Human embryonic stem cells provide info about human development events.
• The goal is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs.
• Medical conditions like cancer and birth defects, are due to abnormal cell division and differentiation.
• A complete molecular controls on these processes suggests new therapies, and controlling these mechanisms helps require additional specialized signals regulate division.

Advantages of embryonic stem cells over adult stem cells

• Embryonic Stem Cells (ESCs) are pluripotent and can become any cell in the human body.
• Adult Stem Cells
  • Adult Stem Cells (ASCs) are Multipotent- can turn into only some cells.
  • ASCs Blood stem cells develop into blood cells only, brain, kidney or liver
• ESCs are have stable cell divisions while ASCs capacity for cell-renewal is limited
• ESCs are easy to obtain but blastocyst is destroyed while ASCs difficult to isolate in its source.

Induced Pluripotent Stem Cells (iPS cells)

• "Reprogram" cells with a specialized function (for example, skin cells) in the laboratory => exhibit similar morphology and growth properties as ES cells and express ES cell-specific genes.
• Created by inducing the specialized cells to express genes - Oct3/4, Sox2, Klf4 and c-Myc that are important for maintaining the defining properties of embryonic stem cells.
• Embryonic stem cells and iPS cells share many characteristics, including the ability become the cells of all organs and tissues, but can sometimes behave slightly differently.
• c-Myc induces cellular immortality and open chromatin structure, while Klf4 suppress apoptosis.
• Oct3/4 changes cell fate from tumor cells to pluripotent cells.
• Sox2 is necessary to establish pluripotency.
• iPS cells were originally established by the delivery of transgenes by MMLV (Moloney murine leukemia virus)-based retroviral vectors to deliver the transgenes
• After constant transgene expression. comes Inactivation of the retroviral promoter by DNA modification.
• Expression of retroviral transgenes is gradually suppressed during reprogramming and somatic cell silencing.
• Viruses are currently used to introduce the reprogramming factors into adult cells, and viruses used to introduce the stem cell factors sometimes causes cancers
• Mouse iPSCs demonstrate properties for pluripotent stem cells but human iPSC need 2 weeks while mice need two weeks
• problems include the low efficiency of establishing iPS cell lines and several variations that raises the concern about pluripotency.

Induced Pluripotent Stem Cells and the iPS-derived Mouse

• Isolation 1 of fibroblasts from mice and induced, by transfection, several cells.
• The cells were then identified and were applied to the next-gen live embryos as cells that had good drug resistance were separated.
• Stable, induced pluripotent stem colonies that were then identified with normal bone marrow cells.
• Offspring carried original content.
• Most iPS cell lines established in earlier studies were derived from fibroblasts.
• iPSc, several different groups shows can be generated for cell types as neuronal progenitor cells, keratinocytes, hepatocytes.
• The efficiency varies among several cell types where human keratinocytes from skin biopsies can be re programmed to much higher and faster fibroblasts.
• Different is given from higher-levelous level c-Myc and Klf4 than fibroblasts which conversion and conversion through cell reprogramming in fibroblasts.
• iPSCs are nearly identical which should give the least amount for rejection of the immune system.
• iPS cells make the research method by creating cells that fight specific diseases by cellular research.

Risk of iPS cells

• It must be determined if I P S are matured for it's research.
• The most noted problem is the virus for it to be treated safely in future procedures.
• More importantly these cells need to be regulated to prevent any type of tumor growth when transplanting is is in progress.
• Overcome the problem by having a general to the four factors to to trigger cellular programming event and not permanent.

Alternative gene delivery strategies used the problem of cell gene delivery by several vectors

• Non-integratable Vector
• DNA Transduction
• Protein Transduction
• Use of small molecules to the pluripotency program which creates factor-free iPS cells
• hESC is a more controlled method for diseases

ESC vs iPSC

• ESCs come from inner cell mass of blastocyst
  • Requires the destruction of human embryos ex utero and are highly ethically controversial
  • Limited supply and are not patient-specific
• Comparatively, iPSCs come from reprogramming of somatic cells
  • iPSCs generally secure
  • Less ethically controversial and can be created, can be easily derived from a variety of genetic background
  • Epigenetically, it is to be known that ES/iPSc's may be refractory.