A second method by which cells can regulate transcription initiation (outside of binding transcription factors to enhancers) is through the control of _____ ______ (define)
- DNA methylation: a biochemical modification of DNA itself in which a methyl (-CH3) group is added to the fifth carbon on the cytosine base in a 5' CpG 3' dinucleotide pair on one strand of the double helix (the "p" in CpG stands for phosphate
Enzymes called ____ ______ ______ catalyze the mehtylation of _______ in CpG dinucleotides
- DNA methyl transferases (DMNTs)
DNA methylation is particularly important to the control of expression of housekeeping genes in vertebrates, though it also plays a role in regulating some _____-type-specific genes. In the human genome, about 70% of the C residues in CpG dinucleotides are methylated. Give two reasons this DNA methylation can alter gene expression heritably without changing the base sequence of DNA
- cell-type-specific genes
- because DNA methylation affects gene transcription
- also methylation patterns are copied during DNA replication
- alter gene expression heritably without changing the base sequence of DNA
- *Methylation is key to an epigenetic phenomenon seen in mammals (including humans) that is called genomic imprinting
Invertebrate animals and unicellular euks have _____ or ____ DNA methylation, while the worm C. elegans and yeasts have ______.
- little or no
- *The info in this section may not be relevant to all euk organisms, but it is very important to human genetics
DNA sequences that may be a few hundred or a few thousand bp long, and within which the frequency of CpG dinucleotide is much higher than that of the rest of the genome
However, unlike the CpG dinucleotides in the rest of the mammalian genome, the C residues in CpG islands are usually ________. When the CpG islands in the vicinity of a gene's promoter are ________, the chromatin is "open" and the gene is transcriptionally ______
What is the reason that CpG islands are usually unmethylated?
- the proteins that activate transcription by binding to CpG islands prevent DNMTs from methylating these islands
- *These transcriptional activators will be found in many cell types if the target gene is a housekeeping gene expressed in most cells
If the _______ are not present, the CpG island becomes methylated. The gene cannot be transcribed because repressors called ______ _____ _____ proteins (MeCPs) bind to methylated CpG islands and close the chromatin structure
- Methyl-CpG-binding proteins
Imprinting control regions (ICRs)
- The unusual phenomenon in which the expression of an allele depends on the parent that transmits it
- sex-specific methylation of certain DNA sequences
Only about ____ of the 25,000 genes in the human genome exhibit imprinting (via RNA-Seq experiments). About half of the 100 are paternally imprinting vs the other half which are maternally imprinting. Explain both concepts
- Maternal imprinting silences (doesn’t express) the allele inherited from the mother and all the mRNA from that gene is made form the paternal allele
- Paternal imprinting silences (doesn’t express) the allele inherited from the father, while the allele from the mother is transcribed
Why is it that a deletion of a paternally imprinted gene could pass without effect from a father to any child?
What happens if a woman is heterozygous for the same deletion
- Because the child's wild-type maternal allele would be expressed.
- However, if a woman was heterozygous for the same deletion 50% of her children would receive the deletion from her
All of thse heterozygous children would have the mutant phenotype (why?)
Deletion of a maternally imprinted gene could pass unnoticed from mother to daughter for many generation (why?)
- because the one intact copy of the gene they inherit from their father is inactive; no gene product can be made, causing the aberrant phenotype.
- because the paternally derived gene copy is always active
If however, the the deletion passed from a man to his children, both the sons and daughters would each have a ___% chance of receiving a deleted paternal allele, and those children would express the ______ phenotype because the intact copy inherited from their mothers is _______
- 50% chance
Angelman Syndrome and Praeder-Willi Syndrome are both associated with _____ _____ on chromosome 15. Angelman syndrome occurs when the deletion is inherited from the _____. Praeder-Willi is inherited when the deletion is inherited from the _____.
- small deletions
- substantiate with info on pg 558-559
Genes may be modified in a manner that does not change the base pair sequence of the DNA, but nevertheless affects gene transcription in a _______ manner. Modifications to genes that alter gene expression without changing the ____ _____ ______ and that are inherited directly through _____ ______ are epigenetic changes
- base pair sequence
- cell divisions
The type of epigenetic change responsible for genomic imprinting is ____-specific DNA methylation of _____ _________ found in specific _____ that are located near the 100-odd _______ genes
- CpG dinucleotides
- ICRs (imprinting control regions)
- imprinted genes
Why are imprints maintained when somatic cells divide by mitosis?
The presence of a methyl group on one strand of a newly synthesized double helix signals DNMT methylase enzymes to add a ______ group to the other strand. Sex-specific methylation of imprinted loci thus generally remains in the _______ cells throughout the life of the individual
- because the pattern of methylation can be transmitted during DNA replication
- methyl group
What guarantees that imprinting is sex specific?
The pedigrees shown required that the patterns of DNA methylation must be reset during meiosis before being passed on to the next generation
The figure shows that the methylations are ______ in the germ-line cells, and sex-specific methylation marks are then ________ during each passage of the gene through the _____ ______ into the next generation
- erased (removed)
- germ line
Some genes are methylated in the ______ germ line; others receive methylation marks in the ______ germ line. For each gene subject to this effect, imprinting occurs in either the maternal or paternal line, never in ______
- maternal germ line
- paternal germ line
- *The molecular differences in the male and female germ line that result in different patterns of methylation are unknown
DNA methylation at ICRs controls the transcription of _____ ______. In contrast with methylation at CpG islands, which always _______ transcription, methylation at ICRs can ______ or ______ imprinted genes. What are two mechanisms of imprinting?
- nearby genes
- activate or deactivate (turn on or off)
- insulators and ncRNA (noncoding RNA)
The ICR contains an _________ whose function is controlled by DNA methylation. An example of this mechanism for ICR function is seen in the ______ imprinted mouse gene Igf2 (for insulin-like growth factor 2). Imprinting at the Igf2 locus works through ________ of an insulator that lies between the Igf2 _______ and its _________
- promoter and enhancer
The nonmethylated insulator on the maternal chromosome is functional, it binds CTCF, a protein whose association with insulators forms ______ in chromatin. As a result, the enhancer on the maternal chromosome cannot activate _______ of the Igf2 gene because it is not in the _____ ______ as the genome's promoter
- same loop
On paternal chromosome, by contrast, the insulator is _______, which _______ it from binding CTCF. Why does the enhancer activate transcription from the Igf2 promoter without a functional insulator?
- because these two elements are now in the same loop
- *Note in this case that even though it is the paternal chromosome that is methylated, it is the maternal allele that is not transcribed (that is, Igf2 is maternally imprinted)
In the vicinity of some imprinted genes, the ICR encodes an ncRNA whose transcription is controlled by a _____ ______. The paternally imprinted insulin growth factor receptor 2 gene (Igfr2), which encodes the receptor for Igf2, provides an example of this imprinting mechansim. How does this work? 4-story)
- CpG isalnd
- pg 560 mid right
For these RNAs, what are the targets, and effects?
In animals, one of the most abundant classes of small RNAs is composed of the ______-RNAs. They are usually ______ regulators of target mRNAs, resulting in the _____ of these mRNAs or preventing them from being ________
- micro-RNAs (miRNAs)
The human genome has close to 1000 genes encoding miRNAs. These genes are transcribed by RNA polymerase II into long primary transcripts called _______ that contain one or more ______ sequences in the form of mostly ______-stranded stem loops
- double-stranded stem loops
The pri-mRNAs need to be ______ to form the active miRNAs, which are _____ and _____-stranded. This multistep process, which is aided by two ribonuclease enzymes called _____ and _____
- short and single-stranded
- Drosha and Dicer
During the process, the miRNA sequences are transported out of the _______ (where they were transcribed) into the _______ , where they will act. Furthermore, the miRNAs become incorporated in become incorporated into ribonucleoprotein complexes called _____ _____ _____ complexes
- miRNA induced silencing complexes (miRISCs)
The ribonucleoprotein complexes (miRISCs) containing miRNAs mediate diverse functions depending on particular factors, name 1.
Depending on the extent of sequence complementarity between the miRNA in the complex (called the guide) and the target sequences in mRNA 3' UTRs
A miRISC whose guide miRNA has perfect complementarity with the target RNA causes mRNA ______. With less complementarity, the mechanism is usually ______ of translation, although exactly how miRISCs regulate transnational activity is not yet understood
Small interfering RNAs (5-story)
- Unlike miRNAs, siRNAs come from double stranded RNA with 3’ overhangs.
- Like miRNA it is processed by dicer and incorporated into the RISC complex.
- The mRNA is then sliced by the complex.
- Unlike miRNAs, the siRNAs must be perfect matches to the mRNA.
- siRNAs also associate with the RNA Induced Transcriptional Silencing complex (RITS) which recognizes the DNA of sequences which have produced double stranded RNA and silences them through histone modification
Male and female flies exhibit many ____-specific differences in morphology, biochemistry, behavior, and function of the germ line. Through decades of work, researchers concluded that in Drosophila, it is the number of ___ chromosomes, not the presence of the ___, that determines sex, and that sex determination first occurs through ________ regulation of the Sxl gene
- transcriptional regulation
Transcription of Sxl in XX (and not XY) animals initiates a cascade of events that influences sex through three independent pathways: (Name them)
pg 564-565 top right
An understanding of the 1st pathway mentioned emerged from analyses of ________ that affect particular ______ characteristics in one sex or the other
In early embryogenesis (before sex determination and dosage compensation have taken place), XX cells transcribe Sxl from the ______ ______. Transcription from ____ depends on four ________ _____ ________: Scute, Runt, SisA, and Upd
- "establishment promoter" (Pe)
- transcriptional activator proteins
Why do XX embryos have twice as much of these four activators as XY embryos?
- because the genes for these activators are on the X chromosome
- *Only in cells with two X chromosomes is the concentration of activators sufficient for Sxl transcription to occur
Sxl is an RNA-binding protein that controls the ________ _______ of specific RNA targets, including its ____ RNA. As embryogenesis progresses, the transcription factors that activate Sxl transcription from Pe _______, and Sxl is transcribed instead from the ________ _________
- alternative splicing
- maintenance promoter (Pm)
In males, splicing of the primary Sxl transcript produced from the maintenance promoter generates an RNA that includes an ______ containing a stop codon in its reading frame. As a result, this RNA in males is not _______ (explain)
- exon (exon 3)
- reading frame
- productive (it does not generate any Sxl protein)
In females, however, the Sxl protein previously produced by transcription from the ______ ________ influences the _______ of the primary transcript initiated at the ________ ________. When the earlier-made Sxl protein binds to the later-transcribed RNA, this binding alters ______ so that ________ is no longer part of the final mRNA
- establishment promoter Pe
- maintenance promoter Pm
- exon 3
Without exon 3, the mRNA can be translated to make _____ Sxl protein. Thus, a small amount of Sxl protein synthesized very early in development establishes a _______ ______ _____ that ensures more synthesis of Sxl protein in development
- positive feedback loop
The Sxl protein influences the ______ of RNAs transcribed not only from its own _____, but also from other ______. Among these is the transformer (tra) gene. In the presence of the Sxl protein (as in normal females), the tra primary transcript undergoes productive _______ that produces an mRNA translatable to a _______ protein. In the absence of Sxl protein ( as in normal males), the splicing of the tra transcript results in a ________ protein
The Tra and Tra2 proteins also influence the splicing of doublesex (dsx) gene's primary transcript. This splicing pathway results in the production of a ______-specific Dsx protein called Dsx-F. In males, where there is no ____ protein, the splicing of the dsx primary transcript produces the related but different _______ protein. The N-terminal parts of the Dsx-F and Dsx-M proteins are the ______, but the C-terminal parts of the proteins are _______
- female-specific Dsx protein
- Dsx-M protein
Although both Dsx-F and Dsx-M function as _______ factors, they have _______ effects. In conjunction with the protein encoded by the intersex (ix) gene, Dsx-F primarily _______ the transcription of genes whose expression would generate the _______ ______ characteristics of _______. However, it also activates the transcription of genes that promote ______ ______
- somatic sexual
- somatic femaleness
Dsx-M, which works independently of the Intersex protein, does the ______; it is primarily a transcriptional activator of ______ genes, and it also ________ femaleness genes
Interestingly, the two Dsx proteins can bind to the same _______ elements, but their binding produces opposite outcomes (3-story)
pg 567 left
State the plant branch providing the pollen and the phenotype