Lec 5/6: Epigenetic Mechanisms of gene regulation Flashcards
(175 cards)
1
Q
what are the 2 components of the epigenome?
A
1) Histones associated with DNA
2) DNA methylation- covalently bound to genome, stable long-term signal
2
Q
what is DNA methylation?
A
methyl marks added to certain DNA bases repress gene activity
- covalently modification of cytosine in C-pyruvate- G sequence
3
Q
what is histone modification?
A
combo of different molecules can attach to the “tails” of proteins called histones.
- alter activity of DNA wrapped around them
4
Q
what is the end product of DNA methylation?
A
5-methyl cytosine
5
Q
_____ first discovered methylated DNA in 1948
A
Rollin Hotchkiss
6
Q
what did Hotchkiss discover in experiments of DNA methylation?
A
DNA from certain sources had a 5th base group = 5-methyl cytosine
7
Q
(1970s) Harold Weintraub & colleagues noticed active genes are ___ or _____
A
low in methyl groups
under methylated
8
Q
conclusion that Weintraud made
A
methylation helped repress genes
9
Q
genes associated with high levels of chromatin have ____ levels of methylations
A
lower level of methylation
10
Q
DNA methylation is chemical ____ of DNA
A
modification
11
Q
DNA methylation can be inherited without ____ ____
A
sequence change
- able to fit into epigenetic paradigm
12
Q
which species is DNA methylation common in?
A
30%- plants
10%- vertebrates
- most fungi
13
Q
which species is DNA methylation absent from?
A
yeast
flies
nematodes
14
Q
at what positions on the gene does DNA methylation occur?
A
5’- CG- 3’ (CpG) positions
15
Q
in DNA methylation, high frequency of __ dinucleodites aka ____
A
CG dinucleotides
aka CpG islands
16
Q
what are CpG islands?
A
- typically 300-3000 base pairs in length
- hypomethylated
- near approx 70% human promoters
- methylation correlated with tissue-specific gene expression
17
Q
methylation of cytosine is regulated by…
A
DNA methyltransferase (DMNT)
18
Q
how does DMNT work?
A
deoxycytidine + SAM
–> (DMNT) —>
5-methyl-cytidine + SAH
[cytosine–> 5-methylcytosine]
19
Q
measure levels of SAM and SAH to look at ???
A
levels of methylation
levels of DMNT
20
Q
CG nucleotides predominantly found where?
A
within major grooves
21
Q
blocking Tc by CG groups
A
- through major groove
- can block Tc factors from binding
- methyl groups need to be removed for Tc to bind
22
Q
cytosine methylation maintains ____ ____ ____
A
inactive-condensed chromatin site
23
Q
steps to cytosine methylation
A
Tc + RNA polymerase
–> transcription, acetylation
–> DNA methyltransferase, 5-methyl-C
–> methyl- CpG binding proteins associated with co-repressors, histone deacetylase
–> transcription blocked, deacetylation
–> chromatin compaction
transcriptional silencing
24
Q
what is the association of DNA methylation with compacted heterochromatin?
A
- gene expression is silenced
- active part of chromatin, the acetylation is removed, law down acetylation into histones
- to shut down, need HDAC and presence of DNA methylation = compaction of chromatin
25
active genes are associated with histone ___ and DNA _____
ACTIVE
histone acetylation
DNA demethylation
26
repressed genes are associated with histone ____ and DNA ____
REPRESSED
histone deacetylation
DNA methylation
27
describe the distribution of cytosine methylation in mammals, how do we examine this?
- associated with heterochromatic region s
| - use cytogenetics, heterogeneity visible at cytogenetic scale
28
DNA methylation and histone modifications help to ____ (role in epigenetics)
compartmentalize the genome into domains of different transcriptional potentials
29
(high/low) histone acetylation of
euchromatin vs heterochromatin
euchromatin= high histone acetylation
heterochromatin= low histone acetylation
30
DNA methylation
euchromatin vs heterochromatin
eu= low
hetero= dense
31
what type of methylation in euchromatin vs heterochromatin
eu= H3-K4 methylation
hetero= H3-K9 methylation
- methylation can be associated on the protein level, there is relationship between protein tails and target
32
DNMT3L is a protein closely (structurally) related to ___ and _____
DNMT3A and DNMT3B
33
what is the known function of DNMT3L
support protein for functions of DNMT3A and DNMT3B
34
what is the function of DNMT1?
replication of methylation sequences from mother to daughter during cell division
- maintenance of methylation
- inactivity associated with cancer cells
35
DNMT3A/3B associated with what type of methylation?
De Novo methylation
- occurs after cells divided, adding methylation groups
36
Name the 2 types of DNA methylation and which DNMT groups associated with each
maintenance methylation = DNMT1
De novo methylation= DNMT3A, 3B
37
what is maintenance methylation/
methylation of newly synthesized DNA strand at positions opposite methylated sites on parent strand
- after DNA replication
- DNMT1 will sit at point of replication and scan across sequence of newly synthesized DNA, copying methylation group of opposite strand
38
what is De Novo methylation?
- methylation of new positions
| - changes the pattern of methylation in a localized region of the genome during gametogenesis and early development
39
sodium bisulfite sequencing is a method of .....
DNA methylation analysis
40
what occurs during sodium bisulfite sequencing?
- methylated cytosine is unaffected
- converts unmethylated cytosine to uracil
- during PCR(converts U-T) and sequencing, the ration of C and T present at CpG sites is quantified, reflecting methylation level of that site in genomic DNA
41
sodium bisulfate converts....
PCT converts....
C--> U
C--> T
42
what is the difference between sodium bisulfate and ChIP?
??
43
what happens to methylated DNA if treated with sodium bisulfate?
they are protected from conversion (no C-->U)
44
name 3 biological functions of DNA methylation
- transcriptional regulation of 'cellular' genes
- role in mammalian development (imprinting)
- heterochromatin formation
45
why is imprinting important in mammalian development?
DNA methylation is important process for inheritance of gene expression
46
why is heterochromatin formation important in DNA methylation?
silencing gene expression
47
what is the PWWP domain named after?
Pro-Trp-Trp-Pro motif
48
what are PWWP domains?
- domains on the DNMT3A and DNMT3B enzymes
- target DNA methyltransferase activity to heterochromatin
- important to recognize binding to methylated DNA
- have to be recruited by Tc
49
what 2 types of RNA are important for shutting down regions of chromatin material?
RNA-I & non-coding RNA
50
what is a MBD?
methyl-CpG- binding protein
51
what is the function of MBD?
help recruit other larger complexes and HDAC
52
readers vs writers?
readers= DNA methyl binding proteins (MBD)
writer= DNMT
53
what is MECP2?
methyl-CpG-binding protein 2
- if the gene encoding this protein is mutated= Rett syndrome (mental retardation)
54
name 6 characteristics of MECP2
- transcription repressor
- X-linked
- able to bind single methyl- CpG
- binds tightly to chromosomes, heterochromatin
- associates with HDAC complex
- expression correlates with maturation of neural system
55
mutations of MECP2 result in (3)
- Rett syndrome (females)
- Angelmann syndrome
- autism
56
what happens if we knock-out MECP2?
no embryonic development
57
why are DNMTs essential? if mutate/KO they result in??
DNMT1= embryonic lethal
DNMT3A= perinatal death
DNMT3B= embryonic lethal
58
the DNA binding proteins will recruit..... which is called??
histone deacetylases
called NuRD complex (nucleosome remodeling deacetylase) complex
- important for deacteylation of chromatin
59
DNA methylation is facilitated by .....
DNMT
60
methyl binding protein facilitate the recruitment of ....
HDAC
61
what is a mediator in methylation induced gene silencing?
methyl-CpG- binding proteins (e.g. MECP2)
62
how is MECP2 involved in induced gene silencing?
- MBD= repressive
- MECP2 (KO mouse= embryonic lethal)
- has methyl CpG binding domain, and transcriptional repression domain
- interacts with mSin3 co-repressor complex which associates with HDAC to repress transcription
63
what is a proposed mechanism (to avoid passive methylation during cell cycle)
stable repression of gene expression through development
64
what is the stable repression of gene expression through development
- Tc= transient (DNA methylation is not)
- Tc pull in specific enzymes involved in epigenetic process
- MECP2 is removed from DNA to facilitate DNA methylation through phosphorylation
65
what occurs when MECP2 is removed from DNA to facilitate DNA methylation through phosphorylation?
- change in orientation & structure= no longer able to bind DNA and get removed
- kinases that phosphorylate MECP2 are important in process of demethylation to get gene expression
66
name the 3 ways to regulate genes with DNA methylation
1) none present- unmethylated promoter allows gene transcription
2) direct mechanism
- methylated CpGs block binding of Tc
- block transcription
3) indirect mechanism
- Me-CpG binding proteins block Tc binding to promoter
67
role of HDAC in indirect mechanism of gene regulation
- methylation of CpG islands upstream of a gene provides recognition signals for MeCP components of HDAC
- HDAC modifies chromatin in region of CpG island= inactivates the gene
** shut down gene expression via recruitment
68
how does drug TSA (trichostatin A) activate gene expression?
- block HDAC activity
- prevent DNA methylation dependent repression
= HDAC inhibitor
- facilitates histone acetylation
69
how does drug sodium butyrate activate gene expression?
- mimic histone acetylation
| - 'loosen up the chromatin'
70
*** compare:
within transcriptionally inactive chromatin/heterochromatin:
1) chromatin conformation
2) DNA CpG methylation
3) histone modification
1) closed chromatin, highly condensed
2) methylated CpG (incl. at promoter regions)
3) histone deacetylated
- methylated H3-K9me
71
*** compare:
within transcriptionally active chromatin/heterochromatin:
1) chromatin conformation
2) DNA CpG methylation
3) histone modification
1) open chromatin, extended conformation
2) unmethylated CpG (especially at promoter region)
3) histone acetylated
- methylated H3-J4me3,
72
name the 7 essential roles of cytosine methylation in mammals
1) gene expression
2) chromosomal stability
3) cell differentiation
4) imprinting
5) X-inactivation
6) carcinogenesis
7) aging
73
what are the 4 differentiating points in epigenetic programming during development?
totipotent (zygote)
pluripotent (blastocyst, embryonic stem cells)
multipotent (neural stem cells etc. ready to be differentiated into particular cells)
unipotent
- inc differentiation
- facilitated through inc in DNA methylation at specific CpG-dinucleotides
74
stages of nuclear transfer (how differentiated cels become totipotent)
- nucleus removed from egg
| - replaced by nucleus from donor cell
75
what is nuclear equivalence?
differentiated cells maintain the potential to generate an entire organism
- methylation profile acts as molecular biomarker from the cells we take the DNA from
76
2 examples involving reprogramming of genome
1) cloning by nuclear transfer (regenerate entire organism from transfer of single nucleus)
2) induced pluripotent stem cells-- expression of 4 genes are sufficient to transform differentiated cells to "Stem cells"
77
describe quantity of nucleosomes in promoter region
diminished amount
- facilitating RNA polymerase must be more accessible for the machinery
- opening and closing is better facilitated with fewer nucleosomes
78
3 main roles DNA methylation plays in mammalian development
- maintenance and inheritance of tissue-specific gene expression **
- inhibition of transposone gene expression
- genomic imprinting
79
what is inhibition of transposone in DNA methylation?
- prevent transposition
- inhibit DNA recombination between repetitive sequences
- lower probability of genome rearrangements
80
role of genomic imprinting in mammalian development
- inactivation by methylation of a gene on one of a pair of homologous chromosomes
81
in which species does parent-to-origin gene expression occur?
genomic imprinting
- marsupials, eutherian mammals
- involved in placenta and nutrient transfer from mother to embryo (not egg)
82
imprints are epigenetic instructions laid down in the ____ ____ cells
parental germ cells
83
____ expressed imprinted genes tend to promote growth
____ expressed imprinted genes tend to suppress growth
paternal= promote
- genes enhance extraction of nutrients from mother during pregnancy
maternal= suppress
- limits nutrient extraction in pregnancy
84
imprinting anomalies manifested as _____ in early development and _____ when altered later in life
early= developmental/neurological disorders
later= cancer
85
what maternal diet during pregnancy alters expression of imprinted genes in offspring
maternal methyl deficient diet
86
describe epigenetic imprinting of autosomal genes with a sex
- unequal expression of maternal and paternal alleles of a gene
- imprinted/marked with their gametic origin
- imprinting results in parent of origin dependent monoallelic expression
- maternal and paternal genomes are not functionally
- have different outcomes depending on which allele is imprinted on the parental genome
87
example of autosomal gene imprinting
insulator model for control of gene expression at h19 and Igf2 imprinted locus
88
describe the example: insulator model for control of gene expression at h19 and Igf2 imprinted locus
maternal= H19 expressed, Igf2 silenced
paternal= H19 suppressed, Igf2 expressed
89
3 phenotypic effects of imprinting
1) in utero effects (insulin & insulin-like growth factors, placental growht)
2) postnatal effects (movement disorders, lactation, brain development/function)
3) genetic disorders (epimutations, Prader-Willi syndrome, Angelmann syndrome)
90
what is Prader-Willi syndrome?
- deletion/inactivation of genes on paternally inherited chromosome 15
- maternal copy is imprinted and silenced
91
what is Angelmann syndrome?
- deletion/inactivation on the maternally inherited chromosome 15
- paternal copy is imprinted and silenced
92
what is a methylome?
all the CpG sites that could be methylated
93
inc or dec in methylation during fertilization?
decreased
- bc reprogramming is occurring
94
cancer results from loss of methylation in which parental genome?
maternal genome
95
changes in DNA methylation during aging in humans occurs when??
- replication errors at cell division
| - also at post-mitotic tissues
96
inc gene expression= more loosely _____ histones
deacetylated histones
97
2 stages in the history of Barr Body
1949: Murray, Barr, Bertram
- dark, drumstick-shaped masses in nuclei in nerve cells
- pattern only seen in cats
1961: Lyon
- Lyon Hypothesis
- condensation of X chromosome mechanism for inactivating the genes on the chromosome
- named inactive X chromosome= Barr body
(when females are inheriting 2X, one has to be inactivated through process of DNA methylation)
98
describe X-inactivation and somatic mosiacism in dosage compensation
genomic imprinting that leads to almost total inactivation of one X in female mammalian cell
- - Xist gene is one of the X chromosomes expressed, leads to inactivation of chromosome
-- Xist gene in 2nd chromosome is inactive (DNA methylation) -- this remains active
mosiac= periods of CpG islands
99
what is the XIC region?
region where Xist is expressed
100
Xist is transcribed from what ???
ONLY from inactive X
101
the XIC gene is responsible for Cis ____
Cis inactivation
- cis= acting on same chromatin material
102
3 steps in how Xist silences the future inactive X
- expressed from the future Xi
- coats the chromosome
- establishes the inactive state
- recruits methyl binding proteins, HDAC, and other machinery to inhibit acetylation to establish inactive state
103
H4 acetylation shows what??
levels of activation with Xist expression and X chromosome inactivation
inc acetylation H4= associated with inactive chromosome
104
what are the sequential epigenetic modifications that occur during inactivation to silence the chromosome?
- expression of Xist RNA to coat the chromatin
| - results in hypo-acetylation & inc methylation of histones
105
do not get stable inactivation of gene expression until????
demethylation occurs
106
evidence of histone code example
- transcriptional silencing of X chromosome correlates with distinct histone modification patterns
e. g. inactive X chromosome shows reduced H3K4-methylation, then inc H3K27-methylation, then dec H4 acetylation
107
histone modification and gene expression are associated with ????
inactivation of chromosome
108
tortoiseshell cat coat colour
- cannot be cloned
- dosage compensation in cats by X-inactivation
- all cells have the same genotype, different copy of X chromosome is expressed in different cells
- random X-inactivation during embryonic development causes patchwork coloured coat in some females
(does not occur in males)
109
name the 3 genetic/epigenetic mechanisms underlying chromatin-related disorders
epigenetic
1) genomic imprinting defects= altered DNA methylation
genetic
2) trans effects (indirect)= chromatin effector mutation
3) cis effects (Direct)= regulatory sequence mutation (e.g promoter)
110
name 3 examples of gene imprinting defects
- beckwith-wiedemann syndrome
- prader-willi syndrome
- transient neonatal diabetes mellitus
111
name 2 trans effects and examples for each
1) defects in methylation machinery
- systemic lupus erythemtosus
- immunodeficiency, facial anomalies syndrome
- RETT syndrom
2) defects in histone modification enzymes
- Rubinstein-Taybi syndrome
- Coffin-Lowry syndrome
112
name 2 examples of cis-effects that result in disorder/disease
- Fragile X syndrome
- facioscapulohumeral muscular dystrophy
- mutation causing loss in methylation, which is important in stability of genome, lead to inc in inappropriate genes
113
direction of the DNA methylation reaction is modulated by ....
chromatin structure
114
passive DNA demethylation is replication dependent/independent?
replication dependent
115
passive DNA demethylation occurs due to....
occurs where??
lack of maintenance methylation during several cycles of DNA replication
- result in ineffective machinery
- zygotic maternal genome, after fertilization, diving cells (E.g. glial cells)
116
active DNA demethylation is replication dependent/independent?
replication independent
117
active DNA demethylation occurs ....
occurs where?
in the absence of DNA replication
- zygotic paternal genome, after fertilization, non-dividing cells (e.g. neurons)
118
list the 4 pathways for active DNA demethylation in animal cells
1) hydrolytic demethylation
- removes C
2) dinucleotide excision
- cut out CG
3) 5-meC excision
- replaced by DNA repair machinery
4) deamination and thymine excision
- deamination makes C analogous to T
- recognized by DNA repair machinery, substituted for C
119
what is excision?
DNA repair
| - how demethylation occurs
120
what is the deamination reaction?
C--> T --> C
121
what is the DNA demethylation mechanism that involves base excision repair?
- oxidation of 5-methyl-C to create 5-hydroxy-C
- provides target facilitating replace of hydroxy methylation to non-methylated cytosine
- facilated by Tet
122
what is Tet?
- family of dioxygenases (ten elevent translocation)
| - TET family proteins catalyze conversion of 5mC to 5hmC
123
2 mechanisms involved in DNA demethylation?
- DNA methylation
| - hydroxymethylation (conversion 5mC to 5hmC in DNA by TET1)
124
in mammalian genome, where does methylation mostly occur?
carbon-5 position of cytosine reside of CpG dinucleotides
converting C to 5mC
125
compare the promoter region in the C, 5mC, 5hmC
C= promoter is demethylated
5mC= promoter is being silenced
5hmC= promoter on way to gene activation
126
what are the biological functions of 5hmC?
- 5hmC levels are reduced in cancer tissues
- 5hmC containing DNA= enrichment within exons and near transcriptional start sites
- role in transcriptional regulation, contribute to 'poised' chromatin state
- undifferentiated cells: reduction 5hmC associated with inc methylation and cellular differentiation
127
in poised state
inc in 5mC= ___ in 5hmC
inc 5mC= dec 5hmC
128
what DNA demethylation intermediates can Tet proteins generate?
5fC & 5caC
- derived from 5mc
129
describe the new model for DNA demethylation (including 4caC)
- 5mC and 5hmC are oxidized to 5caC by Tet
- 5caC recognized and excised by thymine-DNA glycosylase
- resulting abasic site, induce base excision repair pathway, leading to incorporation of unmethylated cytosines
130
list the 5 steps from 5mC--> C
5mC--> 5hmC--> 5fC--> 5caC---> C
131
what % of mammalian genome encodes mRNA?
2%
132
majority of genome encodes what type of RNA?
- functional long and short non-protein encoding RNA (ncRNA)
133
ncRNA contribute to ???? which all involved epigenetic processes
- transcriptional and post-transcriptional gene silencing
- chromosome dosage compensation
- allelic exclusion
- germ cell reprogramming
- para-mutation
134
ncRNA can initiate _____ through???
initiate gene silencing through covalent modifications of the DNA or its associated histone proteins, interfering with transcription
135
non-coding RNA is also know as?
junk DNA
136
small ncRNA are called???
interference RNA
137
small RNAs control what?
RNAs that code protein
138
small RNAs are a poll of ____ and generally function in ____ ____
pool of 21-24 nt ncRNAs
generally function in gene SILENCING
139
small RNAs contribute to ____ ____ ____ through epigenetic modifications to chromatin
transcriptional gene silencing
140
small RNAs contribute to post-transcriptional gene silencing by _____
affecting mRNA translation or stability
141
important classes of RNA in humans include: (3)
1) endogenous small inferfering RNAs (siRNA, ~700)
2) microRNA (miRNA, ~100)
3) PIWI-interactng RNA (piRNA, possible millions)
142
are are the 2 core components of RNA silencing?
dicers and argonautes
143
explain the process and components of RNA silencing
- double stranded RNA (dsRNA) is processed by Dicer or Dicer-like proteins into short RNA duploexes
- small RNAs associate with ARGONAUTE proteins to confer silencing
144
what are argonaute proteins?
catalytic components of the RNA-induced silencing complex (RISC)
145
what does RISC target for?
gene silencing
146
what are dicer and dicer-like proteins (DCL)?
- in siRNA and miRNA biogenesis, DCL cleave long dsRNA or foldback RNA into 21-25 nt fragments
** first protein we have interaction with
147
benefit of Dicers structure
- allows to measure RNA it is cleaving
| - chops RNA into uniformly-sized pieces
148
what is the second protein we encounter/interact with?
Argonaute
149
what is the function of Argonaute
-after dicer cut up the double stranded RNA, there is association of 21-25 nt RNA with argonaute
- binds small RNAs and their targets
- forms RISC complex (RNA-induced silencing complex)
150
Argonaute proteins named after???
argonaute1 mutatant of Arabidopsis
- argo1 has thin radial leaves, named after octopus it resembles
151
how is siRNA produced?
made by exogenous infection
152
role of siRNA in RNA interference?
- mediated silencing via post-transcriptional an transcriptional gene silencing
- siRNA will be cut into short 21-25 nt length and associated with ARGO protein
- in its single stranded form it will form RISC complex which will bind to complimentary sequences expressed in endogenous cell (to silence the cell)
153
how is miRNA produced?
endogenously
154
role of miRNA in RNA interference?
- mediated slicing of mRNA and translational repression
- able to "knock-down" the gene,
- inhibiting mRNA accumulation
- forms RISC complex, which can target the specific regions of RNA
155
why is RNA interference important?
used in many different organisms to regulate gene expression
- involving small interfering
RNA (siRNA), and microNRAs (miRNA) to silence specific mRNAs in the cytoplasm
156
what % of genome encode RNA sequences that will be used as a template?
only 2% of genome
157
control of gene expression involves the control of ____ ____
transcription initiation
158
gene expression can be controlled after transcription by which 5 mechanisms?
- RNA interference
- alternative splicing
- RNA editing
- mRNA degradation
- protein degradation
159
post transcriptional regulation (6)
1) initiation of transcription
2) RNA splicing (altering the rate of splicing, alternative splicing can produce multiple mRNA from one gene)
3) passage through nuclear membrane
4) destruction of the transcript
5) protein synthesis
6) post-translational modification
160
RNA interference involves the use of ______
small RNA molecules
161
the enzyme ____ chops double stranded RNA into small pieces fo RNA
Dicer
162
2 types of RNA that result from Dicer chopping RNA
1) micro-RNA
| 2) small interfering RNA
163
what is function of micro-RNA (in posttranscriptional regulation)
bind to complimentary RNA to prevent translation
164
what is the function of small-interfering RNA (in posttranscriptional regulation)
degrade particular mRNAs before translation
165
role of introns in posttranscriptional regulation
spliced out of pre-mRNAs to produce the mature mRNA that is translated
166
alternative splicing recognizes....
different splice sites in different tissue types
167
function of mature mRNAS in posttranscriptional regulation
mature mRNAs in each tissue possess different exons, resulting in different polypeptide products from the same gene
168
describe RNA editing in posttranscriptional regulation
- creates mature mRNA that are not truly encoded by the gemone
169
describe the mature mRNA produced from RNA editing
- apolipoprotein B exists in 2 isoforms
- one isoform is produced by editing the mRNA to create a stop codon (prevent further synthesis
- 2 sizes of protein: apoB-100 in liver, B48 in intestine
170
apolipoprotein have a role in ____ ____ in the body
chaperoning cholesterol
171
describe the half-life of mature mRNAs
various half-lifes depending on the gene and location (tissue) of expression
- amount of polypeptide produced from a particular gene can be influenced by the half life of the mRNA molecules
172
how often are proteins produced and degraded in the cell?
continually
173
proteins are labelled with what when degraded?
tagged with ubiquitin
- the protein undergoes conformational change of structure and is targeted towards proteosome for degradation
174
degradation of proteins marked with ubiquitin occurs where????
proteosome
- free enzymes complexes in the cytoplasm
175
what are lysosomes?
membrane-bound vesices sacs (bound by single membrane)
- degradeprotein by endocytosis, phagocytosis and autophagy
